Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point > 9, a pH optimum at 7 and temperature optimum at 50 degrees C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K(m) values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.

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The amino acid sequences of two barley ribosomal proteins, termed HvL17-1 and HvL17-2, were decoded from green leaf cDNA clones. The N-terminal sequences of the derived barley proteins are 48% identical to the N-terminal amino acid sequence of protein YL17 from the large subunit of yeast cytoplasmic ribosomes. Via archaebacterial ribosomal proteins this homology extends to ribosomal protein L22 from eubacteria and chloroplast. Barley L17, and ribosomal proteins L22 and L23 from the archaebacteria Halobacterium halobium and H. marismortui, are 25-33% identical. Interestingly, the barley and archaebacterial proteins share a long, central stretch of amino acids, which is absent in the corresponding proteins from eubacteria and chloroplasts. Barley L17 proteins are encoded by a small gene family with probably only two members, represented by the cDNA clones encoding HvL17-1 and HvL17-2. Both these genes are active in green leaf cells. The expression of the L17 genes in different parts of the 7-day old barley seedlings was analyzed by semiquantitative hybridization. The level of L17 mRNA is high in meristematic and young cells found in the leaf base and root tip. In the leaf, the L17 mRNA level rapidly decreases with increasing cell age, and in older root cells this mRNA is undetectable.

Groups of cDNA clones encoding abundant leaf proteins or derived from genes (gene families) with other features of interest have been selected from a barley leaf cDNA library. The characteriaztion of nine of the groups is summarized and includes information on the tissue specificity and light dependence of expression of their corresponding genes. Different types of control of gene expression are represented in the collection: leaf-specific expression, both stimulated and inhibited by light, constitutive expression, and expression that is maximal in one case in coleoptiles and in two cases in meristematic tissue. For the light-stimulated genes (gene families) encoding chloroplast proteins (Cab, RbcS, and plastocyanin), relative and absolute levels of messengers were determined a5 a Cuuricliuii UC cell age in sections of 7-day-old barley leaves grown under diurnal conditions. Key parameters of cell growth (protein, RNA, and DNA accumulation) were determined in the same leaf sections. The main conclusions of the expression studies are as follows: 1) Light is in no case a requirement for gene expression although it has significant stimulatory effect on some genes: 2) weak expression of some genes coding for chloroplast proteins was detected in the leaf-like, white coleoptiles, whereas expression in roots could not be detected: 3) The cub, rbcS, and plastocyanin genes are expressed very early during leaf cell differentiation, when the plastids morphologically are still in their amyloplast-amoeboid stages: 4) The expression of the cub, rbcS, and plastocyanin genes is not coordinated during leaf cell development.

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