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Type C stay-green mutants are defined as being defective in the pathway of chlorophyll breakdown, which involves pheophorbide a oxygenase (PAO), required for loss of green color. By analyzing senescence parameters, such as protein degradation, expression of senescence-associated genes and loss of photosynthetic capacity, we demonstrate that JI2775, the green cotyledon (i) pea line used by Gregor Mendel to establish the law of genetics, is a true type C stay-green mutant. STAY-GREEN (SGR) had earlier been shown to map to the I locus. The defect in JI2775 is due to both reduced expression of SGR and loss of SGR protein function. Regulation of PAO through SGR had been proposed. By determining PAO protein abundance and activity, we show that PAO is unaffected in JI2775. Furthermore we show that pheophorbide a accumulation in the mutant is independent of PAO. When silencing SGR expression in Arabidopsis pao1 mutant, both pheophorbide a accumulation and cell death phenotype, typical features of pao1, are lost. These results confirm that SGR function within the chlorophyll catabolic pathway is independent and upstream of PAO.

Gene expression for nuclear- and mitochondrial-encoded subunits of respiratory chain components was measured in developing soybean cotyledons and compared to the abundance of the relevant proteins. Overall respiratory gene expression peaked at day 16, close to the peak in cytochrome chain and TCA cycle activities from day 10 to 15. Protein abundance followed transcript abundance for all components examined with the exception of the F1beta subunit of ATP synthase. A dramatic peak in F1beta transcript levels early in development (day 5 to 7) was not mirrored by an increase in protein suggesting translational or post-translational control. Mitochondrial-encoded transcripts were at least 10-fold more abundant than nuclear-encoded transcripts. The pattern of transcript and protein abundance for uncoupling proteins displayed a trend similar to other respiratory proteins examined, implicating similar control mechanisms. The expression of alternative oxidase differed, increasing throughout development with protein peaking at day 20, perhaps suggesting a role in senescence. Overall, this study indicated that respiratory gene expression and protein abundance is co-ordinated with respiratory activity for most components but that some components, such as the F1beta subunit may be under discrete forms of regulation.

Recently, plastidial carbonic anhydrase (CA) cDNA clones encoding functional carbonic anhydrase enzymes were isolated from a 48 h dark-grown cotton seedling (cotyledons) cDNA library (Hoang et al., Plant Cell Physiol. 40: 1999). Here we examined the levels of relative transcript abundance and enzyme activities in cotyledons at different developmental stages and under different environmental conditions (i.e. altering CO2 and light conditions), during post-germinative seedling growth. Relative CA transcript levels and total CA enzyme activity in cotyledons of cotton seedlings increased from 18 h to 72 h of post-germinative growth in the dark, although somewhat later than the glyoxylate cycle enzyme, MS. When 24 h old seedlings were exposed to light for an additional 24 h, CA activity in greening cotyledons increased about 2-fold (compared with controls kept in the dark), whereas relative CA transcript levels were essentially the same. Removal of seed coats from cotyledons of 24 h old seedlings dramatically increased relative CA transcript abundance (measured 24 h later) in the dark, but did not influence CA enzyme activity. Manipulation of external CO2 environments (zero, ambient, or high) modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activity. On the other hand, regardless of the external CO2 conditions, cotyledons of seedlings exposed to light exhibited increased CA activity, concomitant with increased Rubisco activity and increased chlorophyll content. Collectively, our data suggest that steady-state levels of CA and rbcS transcripts are increased in response to environmental CO2 conditions, while CA (and Rubisco) enzyme activities are likely modulated at the post-transcriptional level following exposure of seedlings to light, and in parallel with development of functional chloroplasts.

The roles of light and of the putative plastid signal in glycolate oxidase (GLO) gene expression were investigated in tobacco (Nicotiana tabacum cv. Samsun NN) seedlings during their shift from skotomorphogenic to photomorphogenic development. GLO transcript and enzyme activities were detected in etiolated seedlings. Their respective levels increased three- and six-fold during 96 h of exposure to light. The GLO transcript was almost undetectable in seedlings in which chloroplast development was impaired by photooxidation with the herbicide norflurazon. In transgenic tobacco seedlings, photooxidation inhibited the light-dependent increase in GUS activity when it was placed under the regulation of the GLO promoter P(GLO). However, even under these photooxidative conditions, a continuous increase in GUS activity was observed as compared to etiolated seedlings. When GUS expression was driven by the CaMV 35S promoter (P35S), no apparent difference was observed between etiolated, deetiolated and photooxidized seedlings. These observations indicate that the effects of the putative plastid development signal and light on GUS expression can be separated. Translational yield analysis indicated that the translation of the GUS transcript in P(GLO)::GUS seedlings was enhanced 30-fold over that of the GUS transcript in P35S::GUS seedlings. The overall picture emerging from these results is that in etiolated seedlings GLO transcript, though present at a substantial level, is translated at a low rate. Increased GLO transcription is enhanced, however, in response to signals originating from the developing plastids. GLO gene expression is further enhanced at the translational level by a yet undefined light-dependent mechanism.

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

Several lines of evidence have indicated that lipoxygenase enzymes (LOX) and their products, especially 9S- and 13S-hydroperoxy fatty acids, could play a role in the Aspergillus/seed interaction. Both hydroperoxides exhibit sporogenic effects on Aspergillus spp. (Calvo, A., Hinze, L., Gardner, H.W. and Keller, N.P. 1999. Appl. Environ. Microbiol. 65: 3668-3673) and differentially modulate aflatoxin pathway gene transcription (Burow, G.B., Nesbitt, T.C., Dunlap, J. and Keller, N.P. 1997. Mol. Plant-Microbe Interact. 10: 380-387). To examine the role of seed LOXs at the molecular level, a peanut (Arachis hypogaea L.) seed gene, PnLOX1, was cloned and characterized. Analysis of nucleotide sequence suggests that PnLOX1 encodes a predicted 98 kDa protein highly similar in sequence and biochemical properties to soybean LOX2. The full-length PnLOX1 cDNA was subcloned into an expression vector to determine the type(s) of hydroperoxide products the enzyme produces. Analysis of the oxidation products of PnLOX1 revealed that it produced a mixture of 30% 9S-HPODE (9S-hydroperoxy-10E, 12Z-octadecadienoic acid) and 70% 13S-HPODE (13S-hydroperoxy-9Z, 11E-octadecadienoic acid) at pH 7. PnLOX1 is an organ-specific gene which is constitutively expressed in immature cotyledons but is highly induced by methyl jasmonate, wounding and Aspergillus infections in mature cotyledons. Examination of HPODE production in infected cotyledons suggests PnLOX1 expression may lead to an increase in 9S-HPODE in the seed.

The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-beta-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the beta-glucuronidase reporter gene (uidA. Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tuabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed.

The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light-dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of dark-grown seedlings to light resulted in increased levels of both porA and porB transcripts, as well as increased levels of mRNAs encoding other photosynthesis-related gene products, suggesting that they are under a common mode of regulation. Relative levels of the porA and porB transcripts were similar in seedling cotyledons and primary needles of two-month-old pine trees, whereas only porB transcripts were present at a significant level in mature secondary needles of two-year-old trees. Immunoblot analysis showed that the 37 kDa PORA protein was most abundant in dark-grown tissues, decreased dramatically upon exposure to light, but could still be detected at low levels in light-grown seedlings. In comparison, levels of the 38 kDa PORB protein were not significantly changed upon transfer of dark-grown tissues to light. While both PORA and PORB were detected in cotyledons and primary needles, only PORB could be detected in mature needles. Transcripts derived from the three plastid genes, chlL, chlN, and chlB, encoding subunits of the light-independent protochlorophyllide reductase were detected in the cotyledons and stems of dark-grown seedlings, and in mature needles. The highest levels of chlL, chlN, and chlB transcripts were detected within the top one-third of the stem and decreased gradually towards the stem/root transition zone. Correspondingly, the highest levels of light-independent chlorophyll formation took place near the top of the hypocotyl. A similar pattern of expression was observed for other photosynthesis-related gene products, including porA and porB. Our results suggest that many aspects of the light-dependent, tissue-specific and developmental regulation of POR expression first described in angiosperms were already established in the less evolutionarily advanced gymnosperms. However, unlike angiosperms, light is not the dominant regulatory factor controlling porA expression in these species.

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

Two nodule cDNA clones representing genes involved in Alnus glutinosa nitrogen metabolism were analysed. ag11 encoded glutamine synthetase (GS), the enzyme responsible for ammonium assimilation, while ag118 encoded acetylornithine transaminase (AOTA), an enzyme involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found at highest levels in root nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were found at high levels in nodules, confined to the infected cells, suggesting that in nodules of A. glutinosa, citrulline biosynthesis takes place mainly in the infected cells.

Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A)+ RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing.

A cDNA clone for glyoxysomal citrate synthase (gCS) was isolated from a lambda gt11 cDNA library prepared from etiolated pumpkin cotyledons. The cDNA of 1989 bp consisted of a 1548 bp open reading frame that encoded 516 amino acid residues. The deduced amino acid sequence of gCS did not have a typical peroxisomal targeting signal at its carboxyl terminal. A study of expression in vitro of the cDNA and an analysis of the amino-terminal sequence of the citrate synthase indicated that gCS is synthesized as a larger precursor that has a cleavable amino-terminal presequence of 43 amino acids. The predicted amino-terminal sequence of pumpkin gCS was highly homologous to those of other microbody enzymes, such as 3-ketoacyl-CoA thiolase of rat and malate dehydrogenase of watermelon that are also synthesized as precursors of higher molecular mass. Immunoblot analysis showed that the level of gCS protein increased markedly during germination and decreased rapidly during the light-induced transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the level of mRNA for gCS reached a maximum earlier than that of the protein and declined even in darkness. The level of the mRNA was low during the microbody transition. These results indicate that the accumulation of the gCS protein does not correspond to that of the mRNA and that degradation of gCS is induced during the microbody transition, suggesting that post-transcriptional regulation plays an important role in the microbody transition.

A full-length cDNA clone encoding microbody NAD(+)-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD(+)-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.

Three structurally distinct cDNA clones for cytosolic glutamine synthetase (GS1) were isolated from libraries prepared from senescing radish cotyledons. Northern blot analysis showed that transcripts from two of the three genes encoding GS1, Gln1;1 and Gln1;3, accumulated in the cotyledons during both dark-induced and natural senescence. Transcripts from the last gene, Gln1;2, remained at a low level during both processes. Transcripts from all three Gln1 genes accumulated in cotyledons of germinating seeds. We infer from these findings that GS1 enzymes function in both germination and senescence to convert ammonium to glutamine to remobilize nitrogen from source to sink organs. We have also examined the pattern of expression of these genes in different tissues. All three genes are expressed in roots. A large amount of transcripts from Gln1;1 accumulated in hypocotyls. Whereas none were transcribed in flowers. During dark-induced senescence of cotyledons, application of inorganic nitrogen delayed chlorophyll degradation. Inorganic nitrogen enhanced the accumulation of Gln1;1 transcripts, but decreased those of Gln1;3. In contrast, application of glutamine promoted yellowing of cotyledons during the dark treatment, and slightly increased the amounts of transcripts from Gln1;3 but decreased those of Gln1;1. Transcription of the three Gln1 genes appears, therefore, to be differentially regulated in radish cotyledons during senescence and germination.

cDNA clones encoding cysteine proteinases from cotyledons of germinated seeds of Vicia sativa L. have been obtained by means of PCR. Degenerate oligonucleotide primers were designed according to conserved amino acid regions of known cysteine proteinases. The deduced amino acid sequences of the cDNA clones encoding VSCYSPR1 and VSCYSPR2 display strong homology to cysteine proteinases of the so called papain superfamily. Northern analyses revealed developmentally regulated expression of both the mRNAs in germinating seeds. The transcripts were shown to be products of two distinct single genes, each exhibiting structural polymorphisms as exposed in few nucleotide substitutions.