Arabidopsis nudix hydrolase 7 plays a role in seed germination

Arabidopsis nudix hydrolase 7 (Atnudt7) mutants exhibit reduced seed germination phenotype following after-ripening. The role of AtNUDT7 in seeds and during early stages of imbibition was examined. Seeds of Atnudt7-1 and Col-0 following 3 days of imbibition were used to profile changes in NADH- and ADP-ribose pyrophosphohydrolase enzyme activities, expression of nudix family genes closely related to AtNudt7, and AtNUDT7 protein levels. Changes in pyridine nucleotides, phytohormones, reactive oxygen species and poly(ADP-ribose) levels in after-ripened seeds and 1 day after imbibition were also analyzed. Changes in AtNUDT7 gene expression, protein levels and enzyme activities in WT seeds and during early stages of imbibition were correlated. Atnudt7-1 seeds lacked NADH pyrophosphohydrolase activity that led to very high catabolic redox charge. Abscisic acid (ABA) levels were higher in Atnudt7-1 mutant while salicylic acid, gibberellic acid, and reactive oxygen species (ROS) levels were higher in WT seeds. In Atnudt7-1, there was excess ROS accumulation 1 day after imbibition. PAR levels were significantly higher in Atnudt7-1 mutant when compared to WT during imbibition. Based on these observations, we conclude NADH pyrophosphohydrolase activity conferred by AtNUDT7 is important for NAD:NADH homeostasis in seeds. Perturbations to this key redox couple alter ABA and ROS levels in the seeds that in turn lowers germination.     

Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and Ggamma subunits of the signal transducing heterotrimeric G protein

Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.     

Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana

The sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn(2+) and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0. 36 mm. A V(max) of 12. 7 units mg (-1) for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

<Emphasis Type="Italic">Arabidopsis</Emphasis>plants grown in the field and climate chambers significantly differ in leaf morphology and photosystem components

Background  

Plants exhibit phenotypic plasticity and respond to differences in environmental conditions by acclimation. We have systematically compared leaves of Arabidopsis thaliana plants grown in the field and under controlled low, normal and high light conditions in the laboratory to determine their most prominent phenotypic differences.     

A <Emphasis Type="Italic">var2</Emphasis> leaf variegation suppressor locus, <Emphasis Type="Italic">SUPPRESSOR OF VARIEGATION3</Emphasis>, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold

Background  

The Arabidopsis var2 mutant displays a unique green and white/yellow leaf variegation phenotype and lacks VAR2, a chloroplast FtsH metalloprotease. We are characterizing second-site var2 genetic suppressors as means to better understand VAR2 function and to study the regulation of chloroplast biogenesis.     

A rapid and simple method for constructing stable mutants of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

Background  

Acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate A. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of A. baumannii.     

The CaaX specificities of Arabidopsis protein prenyltransferases explain <Emphasis Type="Italic">era1</Emphasis> and <Emphasis Type="Italic">ggb</Emphasis> phenotypes

Background  

Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. This modification, which mediates protein-membrane and protein-protein interactions, is characterized by the covalent attachment of a fifteen-carbon farnesyl or twenty-carbon geranylgeranyl group to the cysteine residue of a carboxyl terminal CaaX motif. In Arabidopsis, era1 mutants lacking protein farnesyltransferase exhibit enlarged meristems, supernumerary floral organs, an enhanced response to abscisic acid (ABA), and drought tolerance. In contrast, ggb mutants lacking protein geranylgeranyltransferase type 1 exhibit subtle changes in ABA and auxin responsiveness, but develop normally.     

Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana

Background  

Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana.     

<Emphasis Type="Italic">Constitutive Expressor of Pathogenesis-Related Genes5</Emphasis>affects cell wall biogenesis and trichome development

Background  

The Arabidopsis thaliana CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in cpr5 plants, which have leaf trichomes that are reduced in size and branch number.     

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next‐generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen‐activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single‐mutant plants showed a wrinkled seed coat or a burst‐out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent‐cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin‐like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst‐out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild‐type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal‐mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.