Lipid biosynthesis in green leaves of developing maize

Successive leaf sections from the base to the tip of rapidly developing leaves of 7-day-old maize (Zea mays var. Kelvedon Glory), grown in the light, utilized acetate for fatty acid biosynthesis in a very divergent manner. Basal regions of the leaf containing proplastids synthesized insignificant proportions of unsaturated fatty acids and appreciable proportions of fatty acids with 20 or more carbon atoms. An increase in the light intensity during incubations with acetate-1-¹⁴C resulted in very little enhancement of either total or polyunsaturated fatty acid biosynthesis in this tissue.     

Effect of light and ontogenetic stage on sink strength in bean leaves

Light (about 3,000 foot-candles) neither increased nor decreased the sink strength of young, rapidly expanding leaves of Phaseolus vulgaris L. cv. Black Valentine, as measured by the comparative rates of import of ¹⁴C-labeled photosynthates by sink leaves in the light versus dark in short term experiments. Although irradiated sink leaves accumulated more ¹⁴C activity, the difference was fully accounted for by photosynthetic reabsorption of respiratory CO₂ derived from substrates translocated to the sink leaves.

Maximum sink strength was attained when the sink leaf reached 7 to 8 cm² in area (9 to 10% of its fully expanded size). Thereafter sink strength declined rapidly and asymptotically to a near zero value at about 45% final area. During this period, however, the rapid decline in translocation was offset by a rapid rise in the photosynthetic rate of the sink leaf, maintaining a near constant relative rate of dry weight increase until the sink leaf had expanded to about 17% of its final area. Although the increasing photosynthetic capacity was associated with a decreasing import capacity, suggesting that the rate of translocation to the sink leaf was controlled by the developing capacity of the sink leaf for photosynthesis, it was not possible to vary the total (true) translocation rate to the sink leaf by varying the photosynthetic rate of the sink leaf in short term light-dark experiments. Despite a high ratio of source to sink in these experiments, no evidence accrued that translocation into young bean leaves was ever sink-limited.

     

The base of the leaf acts as a localized sink for photosynthate in mature barley leaves

The gradients in photosynthetic and carbohydrate metabolism which persist within the fully expanded second leaf of barley ( Hordeum vulgare ) were examined. Although all regions of the leaf blade were green and photosynthetically active, the basal 5 cm, representing approximately 20% of the leaf area, retained some characteristics of sink tissue. The leaf blade distal from the leaf sheath exhibited characteristics typical of source tissue; the activities of sucrolytic enzymes (invertase and sucrose synthase) were relatively low, whilst that of sucrose phosphate synthase was high. These regions of the leaf accumulated sucrose throughout the photoperiod and starch only in the second half of the photoperiod whilst hexose sugars remained low. By contrast the leaf blade proximal to the leaf sheath retained relatively high activities of sucrolytic enzymes (especially soluble, acid invertase) whilst sucrose phosphate synthase activity was low. Glucose, as well as sucrose, accumulated throughout the photoperiod. Although starch accumulated in the second half of the photoperiod, a basal level of starch was present throughout the photoperiod, by contrast with the rest of the leaf. The ¹⁴CO₂ feeding experiments indicated that a constant amount of photosynthate was partitioned towards starch in this region of the leaf irrespective of irradiance. These findings are interpreted as the base of the leaf blade acting as a localized sink for carbohydrate as a result of sucrose hydrolysis by acid invertase.     

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.     

Within-plant variation in induced defence in developing leaves of cotton plants

According to optimal defence theory (ODT) plants should invest in stronger defence in the most valuable parts, such as reproductive or young tissue. Cotton plants are known to express high resistance to herbivore feeding in the developing leaves at the top of the plant after herbivore-damage. Cotton plants also have developing leaves on side shoots from the nodes all along the plant. This allowed us to investigate within-plant distribution of defence between younger tissues at different locations on the plant. We found that all developing leaves showed increased resistance to feeding by larvae of the generalist moth Spodoptera littoralis after herbivore damage to leaves of the plant. An increase in the concentration of secondary compounds, terpenoid aldehydes, was found in developing leaves both at the top of the plant and on the side shoots. However, the resistance was stronger in the top leaves than in the side shoot leaves, showing that there is intra-plant variation in the induced response between different leaves of the same age. After the initial damage, larval feeding mainly occurred on the older, fully developed true leaves. Furthermore, the herbivore-induced resistance in the developing leaves reduced upward movement of feeding larvae on the plant and reduced the feeding on the upper parts of the plant over a period of at least 6 days. The plant thus benefits from defending all developing leaves by directing feeding to older, less valuable leaves and lower parts of the plant. The observed distribution of defence within cotton plants supports ODT.     

Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh.

Microautoradiography was used to follow the translocation pathways of ¹⁴C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed ¹⁴CO₂, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed ¹⁴CO₂, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C central leaf trace - L left leaf trace - LPI leaf plastochron index - R right leaf trace     

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - 4CL 4-coumarate: CoA ligase - CMT S-adenosyl-L-methionine:caffeate 3-O-methyltransferase - FMT S-adenosyl-L-methionine:vitexin 2-O-rhamnoside 7-O-methyltransferase - HPLC high-performance liquid chromatography - IAT uridine 5-diphosphate L-arabinose:isovitexin 2-O-arabinosyltransferase - PAL L-phenylalanine ammonia-lyase     

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.Abbreviations ADC arginine decarboxylase - Dap diaminopropane - DFMA -difluoromethylarginine - MGBG methylglyoxal-bis-guanylhydrazone - MTA 5-deoxy-5-methylthioadenosine - ODC ornithine decarboxylase - PA polyamines - PAO polyamine oxidase - PCA perchloric acid - PLP pyridoxal phosphate - Put putrescine - SAM S-adenosylmethionine - dSAM decarboxylated S-adenosylmethionine - SAMDC S-adenosylmethionine decarboxylase - Spd spermidine - Spm spermine     

Vanillin-hydrochloric acid as a histochemical test for tannin.

Condensed tannin (leucoanthocyanins and catechins) can be demonstrated in fresh plant sections with saturated alcoholic vanillin followed by addition of concentrated HCl. Bright red vanillin-tannin condensates are formed immediately. Preparations may then be made permanent by mounting in a 1:1 mixture of Hoyer's medium and concentrated HCl. Some fading and loss of tannin occurs. The phloroglucinol-HCl test for lignin can also be made permanent with this acidic mountant.     

Domain interaction in cyanobacterial phytochromes as a prerequisite for spectral integrity

Two phytochromes, CphA and CphB, from the cyanobacterium Calothrix PCC7601, with similar size (768 and 766 amino acids) and domain structure, were investigated for the essential length of their protein moiety required to maintain the spectral integrity. Both proteins fold into PAS-, GAF-, PHY-, and Histidine-kinase (HK) domains. CphA binds a phycocyanobilin (PCB) chromophore at a “canonical” cysteine within the GAF domain, identically as in plant phytochromes. CphB binds biliverdin IXα at cysteine24, positioned in the N-terminal PAS domain. The C-terminally located HK and PHY domains, present in both proteins, were removed subsequently by introducing stop-codons at the corresponding DNA positions. The spectral properties of the resulting proteins were investigated. The full-length proteins absorb at (CphA) 663 and 707 nm (red-, far red-absorbing P _r and P _fr forms of phytochromes) and at (CphB) 704 and 750 nm. Removal of the HK domains had no effect on the absorbance maxima of the resulting PAS–GAF–PHY constructs (CphA: 663/707 nm, CphB: 704/750 nm, P _r/P _fr, respectively). Further deletion of the “PHY” domains caused a blue-shift of the P _r and P _fr absorption of CphA (λ _max: 658/698 nm) and increased the amount of unproperly folded apoprotein, seen by a reduced capability to bind the chromophore in photoconvertible manner. In CphB, however, it practically impaired the formation of P _fr, i.e., showing a very low oscillator strength absorption band, whereas the P _r form remains unchanged (702 nm). This finding clearly indicates a different interaction between domains in the “typical”, PCB binding and in the biliverdin-binding phytochromes, and demonstrates a loss of oscillator strength for the latter, most probably due to a strong conformational distortion of the chromophore in the CphB P _fr form. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Diurnal osmotic cycling in cotton leaves as an indicator of source–sink balance

Abstract Diurnal cycling of osmotic potential was studied in leaves of cotton plants (Gossypium hirsutum L.) grown in the field. Osmotic potential was determined by a pressure-volume procedure as the value coinciding with zero turgor. In plants grown under favourable conditions (no water stress or N stress), osmotic potential at zero-turgor measured at midday was initially about 0.3 MPa lower than before dawn, but this cycling disappeared during the season as the number of fruits per plant increased. In water-stressed or N-deficient plants, osmotic cycling was decreased or even eliminated. Across treatments, cycling of osmotic potential occurred only when plants carried at least 560 cm² of leaf area per fruit. The results are interpreted to mean that diurnal cycling of osmotic potential reveals a ‘sink-limited’ condition within the plant.     

Genetical metabolomics of flavonoid biosynthesis in Populus: a case study

Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds.     

The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves

The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.     

Mathematical modeling of monolignol biosynthesis in Populus xylem

Recalcitrance of lignocellulosic biomass to sugar release is a central issue in the production of biofuel as an economically viable energy source. Among all contributing factors, variations in lignin content and its syringyl-guaiacyl monomer composition have been directly linked with the yield of fermentable sugars. While recent advances in genomics and metabolite profiling have significantly broadened our understanding of lignin biosynthesis, its regulation at the pathway level is yet poorly understood. During the past decade, computational and mathematical methods of systems biology have become effective tools for deciphering the structure and regulation of complex metabolic networks. As increasing amounts of data from various organizational levels are being published, the application of these methods to studying lignin biosynthesis appears to be very beneficial for the future development of genetically engineered crops with reduced recalcitrance. Here, we use techniques from flux balance analysis and nonlinear dynamic modeling to construct a mathematical model of monolignol biosynthesis in Populus xylem. Various types of experimental data from the literature are used to identify the statistically most significant parameters and to estimate their values through an ensemble approach. The thus generated ensemble of models yields results that are quantitatively consistent with several transgenic experiments, including two experiments not used in the model construction. Additional model results not only reveal probable substrate saturation at steps leading to the synthesis of sinapyl alcohol, but also suggest that the ratio of syringyl to guaiacyl monomers might not be affected by genetic modulations prior to the reactions involving coniferaldehyde. This latter model prediction is directly supported by data from transgenic experiments. Finally, we demonstrate the applicability of the model in metabolic engineering, where the pathway is to be optimized toward a higher yield of xylose through modification of the relative amounts of the two major monolignols. The results generated by our preliminary model of in vivo lignin biosynthesis are encouraging and demonstrate that mathematical modeling is poised to become an effective and predictive complement to traditional biotechnological and transgenic approaches, not just in microorganisms but also in plants.     

Large-scale analysis of protein phosphorylation in Populus leaves

Protein phosphorylation is a key regulatory factor in all aspects of plant biology; most regulatory pathways are governed by the reversible phosphorylation of proteins. To better understand the role that phosphorylated proteins play in a woody model plant, we performed a systemic analysis of the phosphoproteome from Populus leaves using high accuracy NanoLC–MS/MS in combination with biochemical enrichments using strong cation exchange chromatography and titanium dioxide chromatography. We identified 104 phosphopeptides from 94 phosphoproteins and determined 111 phosphorylation sites including 93 occurring on serine residues and 18 on threonine residues. The identified phosphoproteins are involved in a wide variety of metabolic processes. Among these identified phosphoproteins, 68 phosphorylation sites (72 %) were located outside of conserved domains. The identified phosphopeptides share a common phosphorylation motif of pS/pT-P/D-S/A. These data suggest that the Populus metabolism and gene regulation machinery are major targets of phosphorylation. To our knowledge, this is the first gel-free, large-scale phosphoproteomics analysis in woody plants. The identified phosphorylation sites will be a valuable resource for many fields of plant biology, and information gained from the study will provide a better understanding of protein phosphorylation.