Tissue and subcellular localization of CycD2 and KRPs are dissimilarly distributed by glucose and sucrose during early maize germination.

In maize, immunoprecipitation assays have shown that CycD2;2 interacts with KRPs. However, evidence on CycD2;2 or KRPs localization and their possible interaction in specific tissues is lacking and its physiological consequence is still unknown. This work explores the spatiotemporal presence of CyclinD2s and KRPs, cell cycle regulators, during maize seed germination (18 and 36 h) after soaking on glucose or sucrose (120 mM). CyclinD2s are positive actors driving proliferation; KRPs are inhibitors of the main kinase controlling proliferation (a negative signal that slows down the cell cycle). Cell cycle proteins were analyzed by immunolocalization on longitudinal sections of maize embryo axis in seven different tissues or zones (with different proliferation or differentiation potential) and in the nucleus of their cells. Results showed a prevalence of these cell cycle proteins on embryo axes from dry seeds, particularly, their accumulation in nuclei of radicle cells. The absence of sugar caused the accumulation of these regulators in different proliferating zones. CyclinD2 abundance was reduced during germination in the presence of sucrose along the embryo axis, while there was an increase at 36 h on glucose. KRP proteins showed a slight increase at 18 h and a decrease at 36 h on both sugars. There was no correlation between cell cycle regulators/DNA co-localization on both sugars. Results suggest glucose induced a specific accumulation of each cell cycle regulator depending on the proliferation zone as well as nuclear localization which may reflect the differential morphogenetic program regarding the proliferation potential in each zone, while sucrose has a mild influence on both cell cycle proteins accumulation during germination. Whenever CycD2s were present in the nucleus, KRPs were absent after treatment with either sugar and at the two imbibition times analyzed, along the different embryo axe zones.

The glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and hexokinase interact with cell cycle proteins in maize.

Cyclin/cyclin-dependent kinase (CDK) heterodimers have multiple phosphorylation targets and may alter the activity of these targets. Proteins from different metabolic processes are among the phosphorylation targets, that is, enzymes of central carbon metabolism. This work explores the interaction of Cyc/CDK complex members with the glycolytic enzymes hexokinase 7 (HXK7) and glyceraldehyde-3-phosphate dehydrogenase (GAP). Both enzymes interacted steadily with CycD2;2, CycB2;1 and CDKA;1 but not with CDKB1;1. However, Cyc/CDKB1;1 complexes phosphorylated both enzymes, decreasing their activities. Treatment with a CDK-specific inhibitor (RO-3306) or with lambda phosphatase after kinase assay restored total HXK7 activity, but not GAP activity. In enzymatic assays, increasing concentrations of CDKB1;1, but not of CycD2;2, CycB2;1 or CycD2;2/CDKB1;1 complex, decreased GAP activity. Cell cycle regulators may modulate carbon channeling in glycolysis by two different mechanisms: Cyc/CDK-mediated phosphorylation of targets (e.g., HXK7; canonical mechanism) or by direct and transient interaction of the metabolic enzyme (e.g., GAP) with CDKB1;1 without a Cyc partner (alternative mechanism).

Two cyclin Bs are differentially modulated by glucose and sucrose during maize germination.

Cell proliferation during seed germination is determinant for an appropriate seedling establishment. The present work aimed to evaluate the participation of two maize B-type Cyclins during germination and under the stimulus of two simple sugars: sucrose and glucose. We found out that the corresponding genes, ZmCycB1;2 and ZmCycB2;1, increased their expression at 24 h of germination, but only ZmCycB1;2 responded negatively to sugar type at the highest sugar concentration tested (120 mM). Also, CycB1;2 showed differential protein levels along germination in response to sugar, or its absence. Both CycBs interacted with CDKA;1 and CDKB1;1 by pull down assays. By an immunoprecipitation approach, it was found that each CycB associated with two CDKB isoforms (34 and 36 kDa). A higher proportion of CycB1;2-CDKB-36kDa was coincident to an increased kinase activity in the presence of sugar and particularly in glucose treatment at 36 h of imbibition. CycB1;2-CDKB activity increased in parallel to germination advance and this was dependent on sugar: glucose > sucrose > No sugar treatment. At RAM, CycB1;2 was more abundant in nuclei on Glucose at late germination; DNA-CycB1;2 colocalization was parallel to CycB1;2 inside the nucleus. Overall, results point out CycB1;2 as a player on promoting proliferation during germination by binding a specific CDKB isoform partner and changing its cellular localization to nuclei, co-localizing with DNA, being glucose a triggering signal.

Glucose modulates proliferation in root apical meristems via TOR in maize during germination.

The Glucose-Target of Rapamycin (Glc-TOR) pathway has been studied in different biological systems, but scarcely during early seed germination. This work examines its importance for cell proliferation, expression of cell cycle key genes, their protein levels, besides morphology and cellularization of the root apical meristem of maize (Zea mays) embryo axes during germination under the influence of two simple sugars, glucose and sucrose, and a specific inhibitor of TOR activity, AZD 8055. The two sugars promote germination similarly and to an extent, independently of TOR activity. However, the Glc-TOR pathway increases the number of cells committed to proliferation, increasing the expression of a cell cycle gene, ZmCycD4;2, a putative G1/S regulator. Also, Glc-TOR may have influence on the protein stability of another G1/S cyclin, ZmCycD3, but had no influence on ZmCDKA;1 or ZmKRP3 or their proteins. Results suggest that the Glc-TOR pathway participates in the regulation of proliferation through different mechanisms that, in the end, modify the timing of seed germination.

Maize E2F transcription factors. Expression, association to promoters of S-phase genes and interaction with the RBR1 protein in chromatin during seed germination.

For seed germination, it is necessary to restart the cell cycle, a process regulated at multiple levels including transcriptional control, that is executed by the E2F family of transcription factors. We identified 12 genes of the E2F family in maize that are expressed differentially during the first 28 h post imbibition (HAI). E2Fa/b1;1 and E2Fc proteins were characterized as an activator and a putative repressor respectively, both forming heterodimers with DPb2 that bind differentially to consensus E2F response elements in promoters of E2F target genes. Transcripts of target genes for these transcription factors accumulate during germination; in dry seeds E2Fc protein is enriched in the target promoters and is replaced by E2Fa/b1;1 as germination advances. RBR1 is found in the same promoters in non-imbibed and 28 HAI seeds, when DNA replication has concluded, and transcription of the E2F targets should stop. During germination promoters of these target genes seem to be decorated with histone marks related to relaxed chromatin structure. Therefore, E2Fs appear to occupy their target genes in a context of open chromatin, with RBR1 fine tuning the progression between the phases.

Proliferating cell nuclear antigen associates to protein complexes containing cyclins/cyclin dependent kinases susceptible of inhibition by KRPs during maize germination.

The Proliferating Cell Nuclear Antigen, PCNA, has roles in both G1 and S phases of the cell cycle. Here we show that maize PCNA can be found in cells in structures of a trimer or a dimer of trimer, in complexes of high molecular mass that change in size as germination proceeds, co-eluting with cell cycle proteins as CycD3;1 and CDKs (A/B1;1). Using different methodological strategies, we show that PCNA actually interacts with CycD3;1, CDKA, CDKB1;1, KRP1;1 and KRP4;1, all of which contain PIP or PIP-like motifs. Anti-PCNA immunoprecipitates show kinase activity that is inhibited by KRP1;1 and KRP4;2, indicating the formation of quaternary complexes PCNA-CycD/CDKs-KRPs in which PCNA would act as a platform. This inhibitory effect seems to be differential during the germination process, more pronounced as germination advances, suggesting a complex regulatory mechanism in which PCNA could bind different sets of cyclins/CDKs, some more susceptible to inhibition by KRPs than others.

Glucose and sucrose differentially modify cell proliferation in maize during germination.

Glucose and sucrose play a dual role: as carbon and energy sources and as signaling molecules. In order to address the impact that sugars may have on maize seeds during germination, embryo axes were incubated with or without either of the two sugars. Expression of key cell cycle markers and protein abundance, cell patterning and de novo DNA synthesis in root meristem zones were analyzed. Embryo axes without added sugars in imbibition medium were unable to grow after 7 days; in sucrose, embryo axes developed seminal and primary roots with numerous root hairs, whereas in glucose axes showed a twisted morphology, no root hair formation but callus-like structures on adventitious and primary seminal roots. More and smaller cells were observed with glucose treatment in root apical meristems. de novo DNA synthesis was stimulated more by glucose than by sucrose. At 24 h of imbibition, expression of ZmCycD2;2a and ZmCycD4;2 was increased by sucrose and reduced by glucose. CDKA1;1 and CDKA2;1 expression was stimulated equally by both sugars. Protein abundance patterns were modified by sugars: ZmCycD2 showed peaks on glucose at 12 and 36 h of imbibition whereas sucrose promoted ZmCycD3 protein accumulation. In presence of glucose ZmCycD3, ZmCycD4 and ZmCycD6 protein abundance was reduced after 24 h. Finally, both sugars stimulated ZmCDKA protein accumulation but at different times. Overall, even though glucose appears to act as a stronger mitogen stimulator, sucrose stimulated the expression of more cell cycle markers during germination. This work provides evidence of a differential response of cell cycle markers to sucrose and glucose during maize germination that may affect the developmental program during plantlet establishment.

Modulation of CycD3;1-CDK complexes by phytohormones and sucrose during maize germination.

Maize CycD3;1 associates to CDKA or CDKB1;1 proteins during germination and the complexes formed develop kinase activity. These complexes appear to vary in size as germination proceeds, suggesting association to different sets of proteins. CycD3;1 and associated CDK proteins respond to phytohormones and sucrose. Results revealed a reduction in the CycD3;1 protein amount along germination in the presence of indoleacetic acid (IAA) or abscisic acid (ABA), although in the latter protein levels recover at the end of germination. While the levels of CDKA increase with IAA, they decrease with ABA. Both phytohormones, IAA and ABA, increase levels of CDKB1;1 only during the early germination times. CycD3;1 associated kinase activity is only reduced by both phytohormones towards the end of the germination period. On the other hand, lack of sucrose in the imbibition medium strongly reduces CycD3;1 protein levels without affecting the levels of neither CDKA nor CDKB1;1. The corresponding CycD3;1 associated kinase activity is also severely decreased. The presence of sucrose in the medium appears to stabilize the CycD3;1 protein levels.

Comparative genomics and functional analysis of the NiaP family uncover nicotinate transporters from bacteria, plants, and mammals.

The transporter(s) that mediate uptake of nicotinate and its N-methyl derivative trigonelline are not known in plants, and certain mammalian nicotinate transporters also remain unidentified. Potential candidates for these missing transporters include proteins from the ubiquitous NiaP family. In bacteria, niaP genes often belong to NAD-related regulons, and genetic evidence supports a role for Bacillus subtilis and Acinetobacter baumannii NiaP proteins in uptake of nicotinate or nicotinamide. Other bacterial niaP genes are, however, not in NAD-related regulons but cluster on the chromosome with choline-related (e.g., Ralstonia solanacearum and Burkholderia xenovorans) or thiamin-related (e.g., Thermus thermophilus) genes, implying that they might encode transporters for these compounds. Radiometric uptake assays using Lactococcus lactis cells expressing NiaP proteins showed that B. subtilis, R. solanacearum, and B. xenovorans NiaP transport nicotinate via an energy-dependent mechanism. Likewise, NiaP proteins from maize (GRMZM2G381453, GRMZM2G066801, and GRMZM2G081774), Arabidopsis (At3g13050), and mouse (SVOP) transported nicotinate; the Arabidopsis protein also transported trigonelline. In contrast, T. thermophilus NiaP transported only thiamin. None of the proteins tested transported choline or the thiazole and pyrimidine products of thiamin breakdown. The maize and Arabidopsis NiaP proteins are the first nicotinate transporters reported in plants, the Arabidopsis protein is the first trigonelline transporter, and mouse SVOP appears to represent a novel type of mammalian nicotinate transporter. More generally, these results indicate that specificity for nicotinate is conserved widely, but not absolutely, among pro- and eukaryotic NiaP family proteins.

Synergistic use of plant-prokaryote comparative genomics for functional annotations.

BACKGROUND: Identifying functions for all gene products in all sequenced organisms is a central challenge of the post-genomic era. However, at least 30-50% of the proteins encoded by any given genome are of unknown or vaguely known function, and a large number are wrongly annotated. Many of these 'unknown' proteins are common to prokaryotes and plants. We set out to predict and experimentally test the functions of such proteins. Our approach to functional prediction integrates comparative genomics based mainly on microbial genomes with functional genomic data from model microorganisms and post-genomic data from plants. This approach bridges the gap between automated homology-based annotations and the classical gene discovery efforts of experimentalists, and is more powerful than purely computational approaches to identifying gene-function associations. RESULTS: Among Arabidopsis genes, we focused on those (2,325 in total) that (i) are unique or belong to families with no more than three members, (ii) occur in prokaryotes, and (iii) have unknown or poorly known functions. Computer-assisted selection of promising targets for deeper analysis was based on homology-independent characteristics associated in the SEED database with the prokaryotic members of each family. In-depth comparative genomic analysis was performed for 360 top candidate families. From this pool, 78 families were connected to general areas of metabolism and, of these families, specific functional predictions were made for 41. Twenty-one predicted functions have been experimentally tested or are currently under investigation by our group in at least one prokaryotic organism (nine of them have been validated, four invalidated, and eight are in progress). Ten additional predictions have been independently validated by other groups. Discovering the function of very widespread but hitherto enigmatic proteins such as the YrdC or YgfZ families illustrates the power of our approach. CONCLUSIONS: Our approach correctly predicted functions for 19 uncharacterized protein families from plants and prokaryotes; none of these functions had previously been correctly predicted by computational methods. The resulting annotations could be propagated with confidence to over six thousand homologous proteins encoded in over 900 bacterial, archaeal, and eukaryotic genomes currently available in public databases.

A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism.

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.

A central role for gamma-glutamyl hydrolases in plant folate homeostasis.

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development.

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.

Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase.

5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with k(cat) values of 0.1-1.2 min(-1) and K(m) values for 5-CHO-THF and glutamate of 0.4-5 µM and 0.03-1 mM, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the K(m) values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit.

Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed using wild-type tomato fruit and fruit engineered for high folate content. In wild-type fruit, the expression of genes specifying early steps in folate biosynthesis declined during development but that of other genes did not. In engineered fruit overexpressing foreign GTP cyclohydrolase I and aminodeoxychorismate synthase genes, the expression of the respective endogenous genes did not change, but that of three downstream pathway genes-aminodeoxychorismate lyase, dihydroneopterin aldolase, and mitochondrial folylpolyglutamate synthase-respectively increased by up to 7.8-, 2.8-, and 1.7-fold, apparently in response to the build-up of specific folate pathway metabolites. These results indicate that, in fruit, certain folate pathway genes are developmentally regulated and that certain others are subject to feedforward control by pathway intermediates. Microarray analysis showed that only 14 other transcripts (of 11 000 surveyed) increased in abundance by two-fold or more in high-folate fruit, demonstrating that the induction of folate pathway genes is relatively specific.

FolX and FolM are essential for tetrahydromonapterin synthesis in Escherichia coli and Pseudomonas aeruginosa.

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The DeltatyrA strain was, as expected, prototrophic for tyrosine, whereas the DeltatyrA DeltafolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.

Phytotoxic effects of Sicyos deppei (Cucurbitaceae) in germinating tomato seeds.

The phytotoxic effect of allelochemicals is referred to as allelochemical stress and it is considered a biotic stress. Sicyos deppei G. Don (Cucurbitaceae) is an allelopathic weed that causes phytotoxicity in Lycopersicon esculentum, delaying seed germination and severely inhibiting radicle growth. This paper reports in in vitro conditions, the effects of the aqueous leachate of S. deppei-throughout tomato germination times-on (1) the dynamics of starch and sugars metabolism, (2) activity and expression of the cell wall enzymes involved in endosperm weakening that allows the protrusion of the radicle, and (3) whether abscisic acid (ABA) is involved in this altered metabolic processes. Results showed that S. deppei leachate on tomato seed germination mainly caused: (1) delay in starch degradation as well as in sucrose hydrolysis; (2) lower activity of sucrose phosphate synthase, cell wall invertase, and alpha-amylase; being sucrose phosphate synthase (SPS) gene expression down-regulated, and the last two up regulated; (3) also, lower activity of endo beta-mannanase, beta-1,3 glucanase, alpha-galactosidase, and exo-polygalacturonase with altered gene expression; and (4) higher content of ABA during all times of germination. The phytotoxic effect of S. deppei aqueous leachate is because of the sum of many metabolic processes affected during tomato seed germination that finally is evidenced by a strong inhibition of radicle growth.

6-pyruvoyltetrahydropterin synthase paralogs replace the folate synthesis enzyme dihydroneopterin aldolase in diverse bacteria.

Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.

Maize D4;1 and D5 cyclin proteins in germinating maize. Associated kinase activity and regulation by phytohormones.

We have previously reported the expression of four different maize D cyclins during seed germination and showed that cytokinins and auxins stimulate the expression of every cyclin in a differential way. In this paper we characterize the behavior at the protein level of two of these cyclins, CycD5 and CycD4;1. Antibodies were raised against CycD5;2 (which very likely also recognizes D5;1) and CycD4;1 and Western blot studies demonstrated that neither BA nor indol-3 acetic acid (IAA) stimulate cyclin accumulation during germination, compared with control levels. However, phytohormones, particularly IAA, modify the kinase activity associated to D cyclins preferentially at early hours of germination. The associated kinase moiety to D cyclins appears to be of a Cdk-A type because this protein immunoprecipitates with D cyclins and because kinase activity is strongly inhibited by both olomoucine and also by a peptide corresponding to the carboxy end of a maize kip related protein (KRP) protein. There is thus no correlation between mRNA and protein expression for these maize D cyclins during seed germination, although phytohormones may stimulate a signaling cascade that stimulates activation of protein kinase activity in cyclin-Cdk complexes.