Factors affecting dry weight accumulation in developing barley endosperm

Some factors that may be concerned in determining final grain weight in barley ( Hordeum vulgare L. var. distichum ) have been investigated. Variation in endosperm fresh and dry weight, volume and starch content have been recorded at different stages of grain development between anthesis and harvest-ripeness for two barleys, cvs Kym and Golden Promise, differing in final grain weight. Results were recorded under both field and glasshouse conditions. The results suggest that the higher final dry weight of Kym, in comparison with Golden Promise, is a function of both rate and duration of grain filling. Only at later stages of endosperm development did the differences in volume become significant and the Kym endosperms continued to increased rapidly in volume for two to three days after endosperm volume had reached a maximum in Golden Promise. The rates of starch accumulation in both cultivars were very similar but starch deposition continued in Kym endosperms for four to five days after deposition in Golden Promise endosperms had slowed down.     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

A novel late embryogenesis abundant protein and peroxidase associated with black point in barley grains

Black point of barley grain is a disorder characterised by a brown-black discolouration at the embryo end of the grain. Black point is undesirable to the malting industry and results in significant economic loss annually. To identify proteins associated with barley black point we utilised a proteomic approach with 2-DE to compare proteins from whole grain samples of black pointed and healthy grain. From this comparison two condition-specific proteins were identified: a novel 75 kDa late embryogenesis abundant (LEA) protein and a barley grain peroxidase 1 (BP1) that were specifically more abundant in healthy grain and black pointed grain, respectively. Although LEA protein was less abundant in black pointed grain, LEA gene expression was greater suggesting protein degradation had possibly occurred in black pointed grain. Similarly, the increase in BP1 in black pointed grain could not be explained by gene expression. Western blot analysis also revealed that the identified LEA protein is biotinylated in vivo. The role that each of these proteins might have in black point development is discussed.     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

Apparent transinhibition of peptide uptake in the scutellum of barley grain

The uptake of glycylsarcosine (Gly-Sar) into scutella separated from germinating grains of barley ( Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent K_m and decrease in V_max) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly-Sar.
When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly-Sar by 10 to 45%. The inhibition, studied in detail with leucylleucine and prolylproline, was due to a decrease in V_max. The two peptides having no effect were glycylglycine and D-alanyl-L-alanine which are the only peptides known to date acting as purely competitive inhibitors when present together with the substrate Gly-Sar.
Preincubation with leucine, proline and alanine was not inhibitory, although preincubation with the corresponding dipeptides was. This result, together with the demonstration of intact leucylleucine in the scutella after preincubation with leucylleucine, indicates that the inhibition was caused by the intact peptides.
The results support the notion that in the mixed type inhibition the increase in the apparent K_m is due to competition for the carrier at the outside of the membrane, while the decrease in V_max is due to peptides taken up and binding to the carrier at the inside of the membrane.     

Temperature-induced absorbance changes in developing barley chloroplasts

Absorbance changes on cooling and heating of barley ( Hordeum vulgare L. cv. IB65) chloroplasts greened for 12, 48 and 72 h were investigated to understand the structural changes during biogenesis of chloroplast membranes. Upon cooling the chloroplast suspension from 24 to 8°C, a positive absorbance change occurred at 678, 435 and 495 nm in 12, 48 and 72 h greened chloroplasts. During heating from 24 to 45°C negative absorbance changes were observed with some shifts in positions in different chloroplast preparations and a simultaneous increase in absorbance between 690 and 735 nm. For chloroplasts developed for 12, 48 and 72 h the changes in absorbance on cooling were 3.8, 3.3 and 1.9% at 678 nm, and on heating, 8.9, 8.3 and 4.1% at 680 nm.
The differences in absorbance changes are considered as an indication of variations in the structural organization and composition of developing chloroplasts. The reversibility of the absorbance changes was maximum in chloroplasts greened for 72 h and minimum in chloroplasts greened for 12 h. This would suggest that fully developed chloroplasts have more flexibility towards temperature-induced changes in the membranes.     

Effect of amino acids,growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Léger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Léger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Léger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Léger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Léger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Léger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

Root cortical senescence decreases root respiration,nutrient content and radial water and nutrient transport in barley

The functional implications of root cortical senescence (RCS) are poorly understood. We tested the hypotheses that RCS in barley (1) reduces the respiration and nutrient content of root tissue; (2) decreases radial water and nutrient transport; and (3) is accompanied by increased suberization to protect the stele. Genetic variation for RCS exists between modern germplasm and landraces. Nitrogen and phosphorus deficiency increased the rate of RCS. Maximal RCS, defined as the disappearance of the entire root cortex, reduced root nitrogen content by 66%, phosphorus content by 63% and respiration by 87% compared with root segments with no RCS. Roots with maximal RCS had 90, 92 and 84% less radial water, nitrate and phosphorus transport, respectively, compared with segments with no RCS. The onset of RCS coincided with 30% greater aliphatic suberin in the endodermis. These results support the hypothesis that RCS reduces root carbon and nutrient costs and may therefore have adaptive significance for soil resource acquisition. By reducing root respiration and nutrient content, RCS could permit greater root growth, soil resource acquisition and resource allocation to other plant processes. RCS merits investigation as a trait for improving the performance of barley, wheat, triticale and rye under edaphic stress.     

Role of phosphoenolpyruvate carboxylase in malate production by the developing barley aleurone layer

The pathway of malate synthesis in the developing aleurone layer of barley ( Hordeum vulgare L. cv. Himalaya) was investigated. Malate formation did not occur under anoxia. Labelling with [2‐¹⁴C]acetate showed that the glyoxylate pathway was not a significant source of malate. The partitioning of glycolytic carbon flux at the branchpoint between phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) and pyruvate kinase (PK, EC 2.7.1.40) was studied using [U‐¹⁴C]glucose. It was concluded that in aleurone from maturing, rapidly acidifying grains the flux through the PEPC branch relative to that through PK is 3‐5 times greater than in young aleurone. This increase in flux can be accounted for by a 5‐fold increase in PEPC protein determined by western blotting and in PEPC activity measured in vitro.     

Location of sugars in barley seeds during germination by NMR microscopy

The distribution and fluctuation of sugars in germinating barley seeds were examined by ¹³C nuclear magnetic resonance (NMR) spectroscopy, ¹H-NMR imaging and ¹H-NMR localized spectroscopy in relation to morphology. Maltose, sucrose, fructose and oils were detected in intact imbibed seeds by ¹³C-NMR spectra. During the first 6 d of germination, the maltose content increased and the oil content gradually decreased, whilst the levels of sucrose and fructose remained constant. Sugars were located by ¹H-NMR images and ¹H-NMR localized spectra in the vascular bundle of the seeds as well as in the solubilized endosperm. They were also detected in the shoots. The sugars detected in an 80% ethanol shoot extract were sucrose and glucose, which were located in the vascular bundles but not in the mesophyll cells of the coleoptile. They were also located in the basal part of the shoot, but not above 7 mm from the scutellum. The data suggest that the sugars are primarily transported through the vascular bundles and, at the same time, rapidly incorporated into mesophyll cells in the leaves.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

Isolation and characterisation of developing chloroplasts from light-grown barley leaves

Developing chloroplasts were isolated from the basal region of green barley ( Hordeum vulgare L. cv. Menuet) leaves and their ultrastructure and biochemical composition were compared to those of mature chloroplasts from the tip of the same leaves, using two methods of purification on sucrose and Percoll gradients.
When examined and compared to mature chloroplasts, the developing chloroplasts showed well-developed grana stacks, but these last organelles were 2-fold smaller and contained lower amounts of chlorohylls and polar lipids. Only traces of trans -3-hexadecenoic acid could be detected in phosphatidylglycerol of developing plastids. The protein content of these plastids was higher than in mature plastids and showed an increased proportion of polypeptides linked to P-700 chlorophyll α-protein. The photosynthetic activity of these plastids was about 2-fold lower and their photosystem 1/photosystem II ratio higher than in mature chloroplasts.     

Organic acids promote the uptake of lanthanum by barley roots

Organic acids play an important role in metal uptake by, and accumulation in, plants. However, the relevant mechanisms remain obscure. Acetic, malic and citric acids increased the uptake of lanthanum (La) by barley (Hordeum vulgare) roots and enhanced La content in shoots under hydroponic conditions. Concentration-dependent net La influx in the absence and presence of organic acids yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The saturable component followed Michaelis-Menten kinetics. The K(m) values were similar; however, the V(max) values in the presence of acetic, malic and citric acids were 4.3, 2.8, 1.5-times that of the control, respectively. Enhanced uptake of La by organic acids was mediated mainly, but not solely, by Ca(2+) channels. X-ray absorption spectroscopic techniques provided evidence of La-oxygen environment and established that La(III) was coordinated to 11 oxygen atoms that are likely to be involved in the binding of La(III) to barley roots via carboxylate groups and hydration of La(III).     

Changes in water relations and endogenous abscisic acid content of wheat and barley grains and embryos during development

Abstract. Immature cereal embryo development can be controlled by in vitro culture on media containing ABA, or by media of low osmotic potential. To assess the possible in vivo roles of these factors, endogenous ABA levels and water relations of embryos and grains of wheat ( Triticum aestivum L.) and barley ( Hordeum vulgare L.) were determined during development. ABA concentrations remained consistent with those required to inhibit precocious germination in vitro of early stage embryos but not of more mature embryos. With increasing maturity, a difference in water potential developed between grain and embryo, suggestive of an in vivo role for water status in controlling the development of the embryo.     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann     

Peptide transport by germinating barley embryos: Uptake of physiological di- and oligopeptides

The uptake of a variety of physiological di- and oligopeptides by germinating barley (Hordeum vulgare L.) embryos is described. Peptides as large as pentaalanine can be absorbed. Evidence is presented suggesting the peptides are absorbed intact and subsequently undergo rapid intracellular hydrolysis. Uptake shows stereospecificity. The transport of peptides is generally faster than the transport of amino acids, making it likely that the former could play an important role in the mobilization of the protein storage reserves during germination. The peptide transport system in barley is compared with similar systems from other groups of organisms.Abbreviations Gly-sar glycylsarcosine - Gly-sar-sar glycylsarcosylsarcosine - Gly-sar-sar-sar Glycylsarcosylsarcosylsarcosine     

Diurnal variations of nitrate reductase activity and stability in barley leaves

Diurnal variations of nitrate reductase (NR) activity and stability have been studied in leaves of barley seedlings ( Hordeum vulgare L. cv. Herta) grown in an 8 h light/16 h darkness regime. Stability (decay) of NR was tested both in the extracts and in the plants. In the morning, when the plants were transferred to light, NR activity increased rapidly during the first hour and then remained constant. After the photoperiod, activity decreased rapidly during the first hour of darkness and then remained fairly constant during the rest of the dark period. The high NR activity during the photoperiod was associated with low NR stability both in the extracts and in the plants. On the other hand the low NR activity during the dark period was associated with high stability in the extracts and in the plants.