Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

The origin of aliphatic hydrocarbons in olive oil.

BACKGROUND: There are many substances that can interfere with olive oil quality. Some of them are well characterized, but many others have an unknown origin. Saturated hydrocarbons make an extraordinary complex family of numerous molecules, some of them present naturally in vegetable oils. When major natural saturated hydrocarbons are analyzed by standard chromatographic methods, this complex mixture of saturated hydrocarbons appears as a hump in the chromatogram and is commonly named as unresolved complex mixture (UCM), whose origin remains unknown. RESULTS: In this work we studied the occurrence and the origin of aliphatic saturated hydrocarbons in olive oil. Hydrocarbons were analyzed in olive oil and along the industrial process of oil extraction. We also analyzed n-alkanes and the UCM fraction of hydrocarbons in leaf, fruit and oil from different varieties and different locations, and we also analyzed the soils at these locations. CONCLUSIONS: We conclude that the hydrocarbons present in olive oil do not necessarily have their origin in a contamination during olive oil elaboration; they seem to have a natural origin, as a result of olive tree metabolism and/or as the result of an intake and accumulation by the olive tree directly from the environment during its entire life cycle. © 2017 Society of Chemical Industry.

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L.

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.

γ-Tocopherol methyltransferase from the green alga Chlamydomonas reinhardtii: functional characterization and expression analysis.

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is a very important enzyme in tocopherol biosynthesis in all photosynthetic organisms. In this paper, we present the functional characterization and expression analysis of γ-TMT from the unicellular green alga Chlamydomonas reinhardtii. Recombinant TMT1 enzyme was purified and characterized. The size of TMT1 subunit was estimated as 37 kDa by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in accordance with the predicted molecular size after TMT1 cDNA sequence. Recombinant TMT1 also showed an apparent molecular mass of 37 kDa in its native conformation, suggesting that native TMT1 has a monomeric structure similar to the plant TMTs already characterized. pH and temperature dependence of TMT1 activity were also similar to plant TMTs. Substrate specificity studies showed that Chlamydomonas TMT1 is responsible for the conversion of γ- and δ-tocopherol to α- and ß-tocopherol, respectively. The kinetic properties of Chlamydomonas recombinant γ-TMT activity were studied and γ-TMT1 has a similar affinity for γ- and δ-tocopherol. Promoter sequence analysis and expression analysis by northern blot revealed that tmt1 expression is strongly upregulated by high light and downregulated by low temperature. This regulatory pattern of tmt1 expression supports the idea that γ- and α-tocopherol play specific roles in the adaptation to growth under low temperature and high light stress conditions.

Nucleoside Metabolism Is Induced in Common Bean During Early Seedling Development.

Nucleoside hydrolases (NSH; nucleosidases) catalyze the cleavage of nucleosides into ribose and free nucleobases. These enzymes have been postulated as key elements controlling the ratio between nucleotide salvage and degradation. Moreover, they play a pivotal role in ureidic legumes by providing the substrate for the synthesis of ureides. Furthermore, nucleotide metabolism has a crucial role during germination and early seedling development, since the developing seedlings require high amount of nucleotide simultaneously to the mobilization of nutrient in cotyledons. In this study, we have cloned two nucleosidases genes from Phaseolus vulgaris, PvNSH1 and PvNSH2, expressed them as recombinant proteins, and characterized their catalytic activities. Both enzymes showed a broad range of substrate affinity; however, PvNSH1 exhibited the highest activity with uridine, followed by xanthosine, whereas PvNSH2 hydrolyses preferentially xanthosine and shows low activity with uridine. The study of the regulation of nucleosidases during germination and early postgerminative development indicated that nucleosidases are induced in cotyledons and embryonic axes just after the radicle emergence, coincident with the induction of nucleases activity and the synthesis of ureides in the embryonic axes, with no remarkable differences in the level of expression of both nucleosidase genes. In addition, nucleosides and nucleobase levels were determined as well in cotyledons and embryonic axes. Our results suggest that PvNSH1 and PvNSH2 play an important role in the mobilization of nutrients during this crucial stage of plant development.

The role of reactive oxygen species in signalling from chloroplasts to the nucleus.

The coordination of chloroplast function with the rest of cellular activity requires a continual stream of communication from this organelle to the nucleus. Chloroplasts are major sites of the production of reactive oxygen species (ROS) as either by-products of the reduction of molecular oxygen (O(2)) or its excitation in the presence of highly energised pigments. Such ROS, while potentially damaging to the cell, are also important initiators or transducers of signals from these organelles to the nucleus in response to environmental cues. ROS can initiate such retrograde signalling pathways that trigger either programmed cell death or adjustment to changed conditions. Such different outcomes have implications for the way in which signal transduction by ROS is accomplished and is the subject of this review. In response to mild-stress situations, and as a consequence of their reactivity or because of their containment by cellular antioxidant systems, it is proposed that ROS engage with or initiate signalling at or very near their site of production. In contrast, under more extreme conditions, ROS are proposed to diffuse away from their site of production and consequently elicit a different set of signalling events.

Nuclease and ribonuclease activities in response to salt stress: Identification of PvRNS3, a T2/S-like ribonuclease induced in common bean radicles by salt stress.

The increase in soil salinization due to global climate change could cause large losses in crop productivity affecting, among other biological processes, to germination and seedling development. We have studied how salt stress affects nucleic acid degrading activities in radicles of common bean during seedling development. In radicles of common bean, a main nuclease of 37 kDa and two ribonucleases of 17 and 19 kDa were detected. Saline stress did not alter these three activities but induced a new ribonuclease of 16 kDa. All three ribonucleases are acidic enzymes that were inhibited by Zn. The 16 and 17 kDa ribonucleases are inhibited by guanilates. In the genome of common bean, we have identified 13 genes belonging to the T2 ribonuclease family and that are grouped in the 3 classes of T2 ribonucleases. The analysis of the expression of the 3 genes belonging to Class I (PvRNS1 to 3) and the unique gene from Class II (PvRNS4) in radicles showed that PvRNS3 is highly induced under salt stress.

Biochemical and Molecular Characterization of PvNTD2, a Nucleotidase Highly Expressed in Nodules from Phaseolus vulgaris.

Nucleotides are molecules of great importance in plant physiology. In addition to being elementary units of the genetic material, nucleotides are involved in bio-energetic processes, play a role as cofactors, and are also components of secondary metabolites and the hormone cytokinin. The common bean (Phaseolus vulgaris) is a legume that transports the nitrogen fixed in nodules as ureides, compounds synthetized from purine nucleotides. The first step in this pathway is the removal of the 5'-phosphate group by a phosphatase. In this study, a gene that codes for a putative nucleotidase (PvNTD2) has been identified in P. vulgaris. The predicted peptide contains the conserved domains for haloacid dehalogenase-like hydrolase superfamily. The protein has been overexpressed in Escherichia coli, and the purified protein showed molybdate-resistant phosphatase activity with nucleoside monophosphates as substrates, confirming that the identified gene codes for a nucleotidase. The optimum pH for the activity was 7-7.5. The recombinant enzyme did not show special affinity for any particular nucleotide, although the behaviour with AMP was different from that with the other nucleotides. The activity was inhibited by adenosine, and a regulatory role for this nucleoside was proposed. The expression pattern of PvNTD2 shows that it is ubiquitously expressed in all the tissues analysed, with higher expression in nodules of adult plants. The expression was maintained during leaf ontogeny, and it was induced during seedling development. Unlike PvNTD1, another NTD previously described in common bean, the high expression of PvNTD2 was maintained during nodule development, and its possible role in this organ is discussed.

Relationship between ureidic/amidic metabolism and antioxidant enzymatic activities in legume seedlings.

Ureides are nitrogenous compounds with a special function in some legume under nitrogen fixing conditions, the ureidic legumes. In this group, ureides are the predominant nitrogen transport molecule from nodules to the upper part, whereas amidic legumes use amides as nitrogen transport compounds. In this study, the ureide levels have been analysed in seedlings from four ureidic and four amidic legume plants. It has been found that the differentiation among ureide and amide plants already exists in seedlings during early seedling development, with high levels of ureide and allantoinase activity in cotyledons and embryonic axes from ureide plants. Since ureides have been implicated in the response of plant to several stress, total hydrosoluble antioxidant capacity and the levels of several antioxidant activities have been determined and compared among these two legume groups. The total antioxidant capacity did not follow any differential pattern in cotyledons or embryonic axes for the analysed plants. The levels of superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase in both embryonic axes and cotyledons are statistical different between amide and ureide seedlings, whereas the catalase activity was similar among these groups of plants. We discuss than amides and ureides could follow different strategies to protect against oxidation.

Nucleases activities during French bean leaf aging and dark-induced senescence.

During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions.

Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii.

Homogentisate phytyltransferase (HPT) (EC 2.5.1.-) catalyzes the first committed step of tocopherol biosynthesis in all photosynthetic organisms. This paper presents the molecular characterization and expression analysis of HPT1 gene, and a study on the accumulation of tocopherols under different environmental conditions in the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HPT1 protein conserves all the prenylphosphate- and divalent cation-binding sites that are found in polyprenyltransferases and all the amino acids that are essential for its catalytic activity. Its hydrophobicity profile confirms that HPT is a membrane-bound protein. Chlamydomonas genomic DNA analysis suggests that HPT is encoded by a single gene, HPT1, whose promoter region contains multiple motifs related to regulation by jasmonate, abscisic acid, low temperature and light, and an ATCTA motif presents in genes involved in tocopherol biosynthesis and some photosynthesis-related genes. Expression analysis revealed that HPT1 is strongly regulated by dark and low-temperature. Under the same treatments, α-tocopherol increased in cultures exposed to darkness or heat, whereas γ-tocopherol did it in low temperature. The regulatory expression pattern of HPT1 and the changes of tocopherol abundance support the idea that different tocopherols play specific functions, and suggest a role for γ-tocopherol in the adaptation to growth under low-temperature.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal.

Molecular characterization of PVAS3: an asparagine synthetase gene from common bean prevailing in developing organs.

In common bean, asparagine synthetase (AS; EC 6.3.5.4) is encoded by three members of a multigene family called PVAS1, PVAS2 and PVAS3. Two of these genes, PVAS1 and PVAS2, have been extensively studied, but little is known about PVAS3, remaining unclear whether PVAS3 function is redundant to the other AS or if it plays a specific role in Phaseolus vulgaris metabolism. In this work, we used a molecular approach to characterize PVAS3 expression and to gain some knowledge about its physiological function. We showed that, in contrast to PVAS1 and PVAS2, PVAS3 was expressed in all organs analyzed. Interestingly, PVAS3 was the AS gene most highly expressed in nodules, leaves and pods at the earliest stages of development, and its expression decreased as these organs developed. Expression of PVAS3 parallels the accumulation of AS protein and the asparagine content during the earliest stages of nodule, leaf and pod development, suggesting an important role for PVAS3 in the synthesis of asparagine in that period. Furthermore, PVAS3 was not repressed by light, as most class-II AS genes. Surprisingly, fertilization of nodulated plants with nitrate or ammonium, conditions that induce PVAS1 and PVAS2 and the shift from ureides to amide synthesis, repressed the expression of PVAS3 in nodules and roots. The possible implications of this regulation are discussed.

The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Galpha subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light-responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.