Evidence for symplastic phloem unloading in sink leaves of barley

The pathway of phloem unloading in sink barley (Hordeum vulgare) leaves was studied using a combination of electron microscopy, carboxyfluorescein transport, and systemic movement of barley stripe mosaic virus expressing the green fluorescent protein. Studies of plasmodesmatal frequencies between the phloem and mesophyll indicated a symplastic sieve element- (SE) unloading pathway involving thick-walled and thin-walled SEs. Phloem-translocated carboxyfluorescein was unloaded rapidly from major longitudinal veins and entered the mesophyll cells of sink leaves. Unloading was "patchy" along the length of a vein, indicating that sieve element unloading may be discontinuous along a single vascular bundle. This pattern was mirrored precisely by the unloading of barley stripe mosaic virus expressing the green fluorescent protein. Transverse veins were not utilized in the unloading process. The data collectively indicate a symplastic mechanism of SE unloading in the sink barley leaf.     

Malic acid induction of decarboxylating NADP-malate dehydrogenase synthesis in C3-plant leaves]

The activity of decarboxylating NADP-malatedehydrogenase (E. C. 1.1.1.40) in green ethiolated pea and barley leaves and in green leaves of a pea mutant lacking photosystem II is found to be 3-fold increased after the injection of malic acid into cut plants. Protein synthesis inhibitors depressed malic acid-induced increase of the activity of "malic"-enzyme, the effect of chloramphenicol being more pronounced in ethiolated green leaves, and that of cycloheximide--in leaves of a mutant with formed photosynthetic apparatus. Possible dependence of malate-induced biosynthesis of "malic"-enzyme on the degree of NADP reduction in chloroplasts is discussed.     

Involvement of the thylakoidal NADH-plastoquinone-oxidoreductase complex in the early responses to ozone exposure of barley (Hordeum vulgare L.) seedlings

A possible implication of the plastid NADH-plastoquinone-oxidoreductase (Ndh) complex in the response against ozone-mediated oxidative stress in barley (Hordeum vulgare L.) leaves was investigated. After a 4 h treatment, exposure of barley seedlings to moderate ozone concentrations produced leaf-age-dependent increases in lipid peroxidation, peroxidase, and Ndh complex activities in the thylakoid membranes. A significant amount and activity of the Ndh complex were detected in mature barley leaves, but not in young barley leaves. In fact, young barley leaves behaved like ndh-deficient leaves in most of the aspects studied. When plants were exposed to photo-oxidative light after ozone fumigation, the recovery of Fv/Fm was lower in young leaves than in mature leaves. Ozone treatment significantly decreased non-photochemical quenching (qN) in young leaves, but not in mature leaves. Mature leaves showed higher levels of the energy (DeltamuH+) dependent (qE) component of qN. Treatment with antimycin A, an inhibitor of cyclic electron flow, increased the decay of qN produced by ozone in young leaves, but not in mature ones. The reduction state of plastoquinone increased after ozone treatment in mature dark-adapted leaves and was strongly quenched by far red light. It is proposed that the function of the Ndh complex helps the maintenance of qN, probably through the poising of the redox steady-state level of the intersystem carriers and then by optimizing the rate of cyclic electron flow. This should constitute an age-dependent early response in barley leaves, by contributing to minimize photoinhibition in the presence of ozone and high light.     

Dehydrophylloquinone, α-dehydrotocopherolquinone and dehydrotocopherols from etiolated maize and barley shoots

Dehydrophylloquinone, α-dehydrotocopherolquinone, α-dehydrotocopherol and γ-dehydrotocopherol have been isolated from etiolated maize and barley shoots and excised etiolated maize shoots that have been exposed to light. They are not present, however, in green maize shoots, spinach leaves and etiolated bean leaves. Demethylphylloquinone was not detected in any of the tissues analysed.     

Nitrogen uptake,assimilation and transport in barley in the presence of atmospheric nitrogen dioxide

Summary Exposure of the leaves of young barley plants to nitrogen dioxide (NO₂) was shown to affect the rate of translocation of N, the form in which it is transported in the xylem stream and the partitioning of N between roots and shoots. Following its entry through the leaves, NO₂ is assimilated by the plant into reduced nitrogenous compounds which accounted for the major increases in plant N content and growth. The various effects of atmospheric NO₂ upon barley seedlings were strongly influenced by nitrate supply to the roots.     

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.     

REDUCTION OF DEHYDRO-L-ASCORBIC ACID IN GREEN LEAVES

1. The capacity of green leaves for absorbing and converting substancesrelated to L-ascorbic acid was investigated, using detachedleaves of various plants, including soybean, pea, barley andspinach.
2. The greater portion of the absorbed dehydroascorbicacid wasrecovered in the leaves as ascorbic acid, minor portionsbeingdiscovered as dehydroascorbic acid and 2,3-diketogulonicacid. The recovery was about 60% of the absorbed dehydro ascorbicacid, in the cases of detached soybean and barley leaves, forinstance, thus suggesting the instability of the substance invivo.
3. The absorption and conversion of ascorbic acid and 2,3-di-ketogulonic acid in detached green leaves were also investigated.Most of the absorbed ascorbic acid reappeared in the leaves.On the other hand, no evidence for the conversion of 2,3-diketogulonicacid into ascorbic and dehydroascorbic acids was observed. Thegreater portion of the absorbed 2, 3-diketogulonic acid seemsto be decomposed in the leaves, under the condition of the presentstudy.

(Received April 17, 1961; )     

Expression of the Plastid ndhF Gene Product in Photosynthetic and Non-Photosynthetic Tissues of Developing Barley Seedlings

A fragment of the NDH-F subunit of the plastid NAD(P)H dehydrogenasecomplex (NAD(P)H-plastoquinone-oxidoreductase) from barley wasexpressed as a fusion protein in Escherichia coli and an antibodyto the fusion protein was prepared. Western blot analysis usingthe anti-NDH-F antibody showed specificity towards a plastidpolypeptide of approximately 70 kDa present in both photosyntheticand non-photosynthetic barley tissue. The polypeptide was foundin thylakoid membranes of green leaves whereas in etiolatedleaves it was shown to be associated with the membrane fractionof etioplasts. NDH-F levels were higher in roots and etiolatedtissue than in greening or young leaves. During leaf ontogeny,NDH-F levels decreased from young to mature tissue but increasedduring senescence. The accumulation of NDH-F in thylakoids ofyoung leaves was stimulated by photooxidative treatment. Theresults indicate a high degree of expression of plastid ndhgenes (which encode NAD(P)H dehydrogenase sub-units) in non-photosyntheticplastids and under conditions which impair the photosyntheticactivity of chloroplasts. In addition to its putative implicationin photosynthetic electron transport, a non-photosynthetic role,such as chloro-respiration, is proposed for the plastid NAD(P)Hdehydrogenase complex. (Received May 20, 1997; Accepted October 8, 1997)     

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.     

Occurrence and Heterogeneity of Chymotrypsin Inhibitors in Vegetative Tissues of Barley

Inhibitors of chymotrypsin and the alkaline proteinase of Aspergillus oryzae were present in the shoots of barley seedlings and weak activities were also detected in the shoot tops of 6-week-old plants. Treatments which induce inhibitor formation in tomato and potato leaves had no effect when tested on mature leaves, seedlings, or young tillers of barley. Fractionation experiments with isoelectric focusing showed that the barley leaves contained several proteinase inhibitors acting on both chymotrypsin and the Aspergillus proteinase, and one inhibitor which acted only on the Aspergillus enzyme. All of these inhibitors were different from the five Aspergillus proteinase inhibitors which are abundant in the endosperm of resting seeds. Two chymotrypsin inhibitors with weaker activity on the Aspergillus proteinase were present in rootlets and also in embryos of resting seeds. These inhibitors were different from both the endospermal inhibitors and the inhibitors present in young leaves.     

Red young leaves have less mechanical defence than green young leaves

In many plants, leaves that are young and/or old (senescent) are not green. One adaptive hypothesis proposed that leaf color change could be a warning signal reducing insect attack. If leaf coloration involves less herbivory, it remains unclear why leaves in many species are constantly green. To examine whether green leaves reduce herbivory by physical defense as an alternative to the supposed warning signal of red leaves, we conducted comparative analyses of leaf color and protective tissues of 76 woody species in spring. The protective features (trichomes, enhanced cuticle and multiple epidermis) and the distribution of red pigments within leaves were examined in both young and mature leaves. We observed that redness was more frequent in young leaves than in senescent leaves. Compared to 36 species with red young leaves, 40 species with green young leaves showed a significantly higher incidence of enhanced cuticle and trichomes in both phylogenetic and non‐phylogenetic analyses. The phylogenetic analysis indicated that the multiple origins of mechanical protection were generally associated with loss of red coloration. Our finding of relatively poor mechanical protection in red young leaves provides additional evidence for the adaptive explanation of leaf color change.     

Spring versus autumn leaf colours: Evidence for different selective agents and evolution in various species and floras

Red colouration is common in young and old leaves of broadleaf woody species. Assuming that leaf colours are adaptive, we examined, by comparing the colouration in young versus old leaves, the possibility that different selection agents may have operated on spring versus autumn leaf colouration. We observed spring versus autumn colouration in three very different woody floras (Finland, Japan and Israel) in order to allow for a broad ecological and evolutionary spectrum. The null hypothesis was that if the same selective agents operated in spring and autumn, it is expected that when spring leaves are red, they should always be red in autumn, and when spring leaves are green, they should be green or yellow in autumn. We found that green spring leaves are almost exclusively associated with yellow leaf colour at senescence in autumn. Species with red autumn leaves almost always have at least some red colouration in their spring leaves. However, about half of the species with red spring leaves have yellow autumn leaves. Brown autumn leaves were not common in the species we studied. As about half of the species with red spring leaves have yellow autumn leaves but not vice versa, we conclude that there are many cases in which the selecting agents for spring versus autumn leaf colour were not the same.     

Photosynthetic pigment concentration, organization and interconversions in a pale green Syrian landrace of barley (Hordeum vulgare L., Tadmor) adapted to harsh climatic conditions

Tadmor is a Syrian barley landrace that has adapted to semi-arid environments. Its leaves are pale green because of a 30% decrease in the chlorophyll and the carotenoid content of the chloroplasts (leading to a 7·5% decrease in light absorption) compared with barley genotypes that are not adapted to harsh Mediterranean climatic conditions (e.g. Plaisant). This difference in pigment content was attenuated during growth of the plants in strong light, but was strongly amplified when strong light was combined with a high growth temperature. The low pigment content of Tadmor leaves was not associated with significant changes in the pigment distribution between the photosystems or between the reaction centres of the photosystems and their associated chlorophyll antennae. No significant difference in the photosynthetic activity (O₂ production per unit absorbed light) was observed between Tadmor and Plaisant. The conversion of violaxanthin to zeaxanthin in strong light and its reversal in darkness were much faster and operated at a higher capacity in Tadmor leaves compared with Plaisant leaves, resulting in an increased photostability of photosystem II in the former leaves. The accelerated xanthophylls interconversion in the Syrian landrace was associated with, and possibly related to, an increased fluidity of the thylakoid membranes. The lipid peroxide level was lower in Tadmor compared with Plaisant. In contrast, no difference was found in the non-photochemical quenching of chlorophyll fluorescence between the two barley genotypes. The data indicate that the pale green Syrian landrace is equipped to survive excessive irradiance through a passive reduction of the light absorptance of its leaves, which mitigates the heating effects of strong light, and through the active protection of its photochemical apparatus by a rapid xanthophyll cycling.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Leaf development and the role of NADP-malate dehydrogenase in C3 plants

The activity of NADP-malate dehydrogenase (NADP-MDH) was determined in the developing first leaf of the C₃ plants wheat, barley and pea. Light dependent activation of the enzyme was observed in all three species following rapid extraction and immediate assay. Maximum activity was obtained following extraction from preilluminated leaves and incubation on ice for 45 min in the presence of dithiothreitol. In all three species, maximum activity was obtained in the young leaf 4 days after emergence of the seedling (about 2.5 to 3 moles per milligram chlorophyll per min in wheat and barley, and 6 moles per milligram chlorophyll per min in pea). On a chlorophyll basis there was an approximate five-fold decrease in NADP-MDH activity as the leaf matured. A similar pattern was found for phospho-enolpyruvate carboxylase and NADP-malic enzyme which had maximum activity in younger leaf tissue. Similarly, the activity of nitrate reductase in wheat and barley was high in the young leaf and it rapidly declined as the leaf matured. In contrast, the capacity for photosynthesis was relatively low in the young leaf, reaching a maximum 6 to 8 days after seedling emergence. The pattern of change in activity of phosphoribulokinase, an enzyme of the reductive pentose phosphate pathway, was similar to that of photosynthesis. The results suggest NADP-MDH and phospho-enolpyruvate carboxylase have important function(s) in the young leaf, which are not directly linked to C₃ photosynthesis, and which, in part, may be linked to nitrate assimilation and provision of malate to mitochondria.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - NADP-MDH NADP-malate Dehydrogenase - NADP-ME NADP-malic enzyme     

3-O-methyl-D-galactose residues in lycophyte primary cell walls

Acid hydrolysis of cell wall-rich material from young leaves of the lycophyte Selaginella apoda (L.) Spring yielded substantial amounts of 3-O-methyl-D-galactose (1) in addition to the usual major monosaccharides (glucose, galactose, arabinose, xylose and galacturonic acid). The yield of 1 approximately equalled that of galacturonic acid. Compound 1 was identified as 3-O-methylgalactose by its 1H and 13C NMR spectra, and shown to be the D-enantiomer by its susceptibility to D-galactose oxidase. Compound 1 was detected in acid hydrolysates of the alcohol-insoluble residues from young leaves of all lycophytes tested, both homosporous (Lycopodium, Huperzia and Diphasiastrum) and heterosporous (Selaginella). It was not detectable in the charophyte green algae Coleochaete scutata, Chara coralina or Klebsormidium flaccidum, any bryophytes [a hornwort (Anthoceros), four liverworts and three mosses], or any euphyllophytes [a psilopsid (Psilotum), a horsetail (Equisetum), eusporangiate and leptosporangiate ferns, the gymnosperm Gnetum, and diverse angiosperms]. A high content of 1 is thus an autapomorphy of the lycophytes.