Maximal l4 CO 2-fixation in spinach occurs in the middle of the palisade mesophyll [Nishio et al. (1993) Plant Cell 5: 953], however, ninety percent of the blue and red light is attenuated in the upper twenty percent of a spinach leaf [Cui et al. (1991) Plant Cell Environ. 14: 493]. In this report, we showed that green light drives 14 C0 2-fixation deep within spinach leaves compared to red and blue light. Blue light caused fixation mainly in the palisade mesophyll of the leaf, whereas red light drove fixation slightly deeper into the leaf than did blue light. I4 C0 2-fixation measured under green light resulted in less fixation in the upper epidermal layer (guard cells) and upper most palisade mesophyll compared to red and blue light, but led to more fixation deeper in the leaf than that caused by either red or blue light. Saturating white, red, or green light resulted in similar maximal 14 CO 2-fixation rates, whereas under the highest irradiance of blue light given, carbon fixation was not saturated, but it asymptotically approached the maximal 14 CO 2-fixation rates attained under the other types of light. The importance of green light in photosynthesis is discussed.

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The physiological reason that higher plants are green is unknown. Other photosynthetic organisms utilize pigments that strongly absorb green light; therefore, there must have been natural forces that 'selected' the photosynthetic pigments found in higher plants. Based on previously published data and our recent findings about green light and photosynthesis within leaves (Sun et al.), a specific functional role is described for the primary photosynthetic pigments of higher plants, that were derived from green algal progenitors. The particular absorptive characteristics of chlorophylls a and b appear to perform two contradictory, but necessary functions in higher plants. Firstly, chlorophylls a and b absorb light for maximum utilization under non-saturating conditions, a function that is well understood. Secondly, they can act as protective pigments under over-saturating light conditions, when absorbed light is dissipated as heat. Under such conditions, a significant portion of light can also be efficiently utilized, especially in the bottom portion of the leaf, that is mainly illuminated by green light and not down-regulated. The second function may have been the selective force that gave rise to the extremely successful terrestrial plants, that evolved from green algae.

In situ measurements of 14C-CO2 incorporation into 40-[mu]m paradermal leaf sections of sun- and shade-grown spinach leaves were determined. Chlorophyll, carotenoid, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content in similar 40-[mu]m paradermal leaf sections was also measured. The carbon fixation gradient did not follow the leaf internal light gradient, which decreases exponentially across the leaf. Instead, the 14C-CO2 fixation was higher in the middle of the leaf. Contrary to expectations, the distribution of carbon fixation across the leaf showed that the spongy mesophyll contributes significantly to the total carbon reduced. Approximately 60% of the carboxylation occurred in the palisade mesophyll and 40% occurred in the spongy mesophyll. Carbon reduction correlated well with Rubisco content, and no correlation between chlorophyll and carotenoid content and Rubisco was observed in sun plants. The correlation among chlorophyll, carotenoids, Rubisco, and carbon fixation was higher in shade leaves than in sun leaves. The results are discussed in relation to leaf photosynthetic and biochemical measurements that generally consider the leaf as a single homogeneous unit.

Proteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications. Provisional identity of 30 of these gene products was proposed based upon coincidence of measured mass with that calculated from genomic sequence. Analysis of isolated photosystem II complexes allowed detection and resolution of a minor population of D1 (PsbA) that was apparently palmitoylated and not detected in less purified preparations. Based upon observed ؉80-Da adducts, D1, D2 (PsbD), CP43 (PsbC), two Lhcbs, and PsbH were confirmed to be phosphorylated, and a new phosphoprotein was proposed to be the product of psbT. The appearance of a second ؉80-Da adduct on PsbH provides direct evidence for a second phosphorylation site on PsbH, complicating interpretation of its role in regulation of thylakoid membrane organization and function, including light-state transitions. Adducts of ؉32 Da, presumably arising from oxidative modification during illumination, were associated with more highly phosphorylated forms of PsbH implying a relationship between the two phenomena. Intact mass proteomics of organellar subfractions and more highly purified protein complexes provides increasingly detailed insights into functional genomics of photosynthetic membranes. Molecular & Cellular Proteomics 1:46 -59, 2002.With many genomes completed and many more in the pipeline it is clear that the post-genomic era has arrived. Considerable attention is now being directed toward defining the function of individual gene products and the inter-relationships between them (functional genomics). Proteomics seeks to catalogue the full complement of the gene products of a cell and the effect of development, environment, and disease upon their expression. Mass spectrometry is driving proteomics, most commonly as a tool to identify proteins separated and visualized on two-dimensional gels. However, such strategies are insensitive to low abundance proteins (1, 2), proteins that are not fully represented on two-dimensional gels (for example, some classes of intrinsic membrane proteins) and to subtle changes in covalent modifications that do not appreciably alter isoelectric point or electrophoretic mobility. To address some of these shortcomings, intact mass proteomics has been proposed (3-8).The ideal analysis of any protein includes a mass spectrum of the intact molecule to define the native covalent state and its heterogeneity (3, 4). A versatile procedure has been developed for effective electrospray-ionization mass spectrometry (MS) 1 of intact intrinsic membrane prot...

In situ measurements of 14C-C02 incorporation into 40-pm paradermal leaf sections of sun-and shade-grown spinach leaves were determined. Chlorophyll, carotenoid, and ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) content in similar 40-pm paradermal leaf sections was also measured. The carbon fixation gradient did not follow the leaf interna1 light gradient, which decreases exponentially across the leaf. Instead, the 14C-C02 fixation was higher in the middle of the leaf. Contrary to expectations, the distribution of carbon fixation across the leaf showed that the spongy mesophyll contributes significantly to the total carbon reduced. Approximately 60% of the carboxylation occurred in the palisade mesophyll and 40% occurred in the spongy mesophyll. Carbon reduction correlated well with Rubisco content, and no correlation between chlorophyll and carotenoid content and Rubisco was observed in sun plants. The correlation among chlorophyll, carotenoids, Rubisco, and carbon fixation was higher in shade leaves than in sun leaves. The results are discussed in relation to leaf photosynthetic and biochemical measurements that generally consider the leaf as a single homogeneous unit.

lron nutrient deficiency was investigated in leaves of hydroponically grown sugar beets (Beta vulgaris) to determine how ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression is affected when thylakoid components of photosynthesis are diminished. Rubisco polypeptide content was reduced by 60% in severely iron-stressed leaves, and the reduction was directly correlated to chlorophyll content. The concentration of Rubisco protein in ironstressed leaves was found to be regulated by availability of mRNAs, and CO, fixation by Rubisco was reduced from 45 pmol CO, m-' s-' in extracts from iron-sufficient leaves to 20 pmol CO, m-, s-' in extracts from severely stressed leaves. The rate of CO, fixation was directly correlated to leaf chlorophyll content. Rubisco in iron-sufficient control leaves was 59% activated, whereas in severely stressed leaves grown under the same light, Rubisco was 43% activated. RNA synthesis was reduced by about 50% in iron-deficient leaves, but 16s and 25s rRNA and ctDNA were essentially unaffected by iron stress.

, lllinois 61 801 (J.W.)The potency of various uncouplers for collapsing the lightinduced p H gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane p H gradient (ApH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable ApH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 t o 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the ApH was determined using the empirical relationship between the ApH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin.Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the p H gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.