Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using 3H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca2+ was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo

During biosynthesis of chlorophyll, Mg2+ is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximate to 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called 'ATPases associated with various cellular activities', known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg2+ and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis

A red-shifted chlorophyll.

Chlorophylls are essential for light-harvesting and energy transduction in photosynthesis. Four chemically distinct varieties have been known for the past 60 years. Here we report isolation of a fifth, which we designate chlorophyll f. Its in vitro absorption (706 nanometers) and fluorescence (722 nanometers) maxima are red-shifted compared to all other chlorophylls from oxygenic phototrophs. On the basis of the optical, mass, and nuclear magnetic resonance spectra, we propose that chlorophyll f is [2-formyl]-chlorophyll a (C55H70O6N4Mg). This finding suggests that oxygenic photosynthesis can be extended further into the infrared region and may open associated bioenergy applications

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg 2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins

Relação entre níveis de hemoglobina, concentração de retinol sérico e estado nutricional em crianças de 6 a 59 meses do Estado da Paraíba

OBJETIVO: Analisar a relação entre os níveis de hemoglobina, concentração de retinol sérico e estado nutricional em crianças de 6 a 59 meses de idade do estado da Paraíba. MÉTODOS: Corte transversal, de base populacional, envolvendo 1.108 crianças de 6 a 59 meses de idade, de ambos os sexos, do Estado da Paraíba. As concentrações de hemoglobina foram analisadas em sangue venoso por meio decontador automático, e as de retinol sérico por cromatografia líquida de alta resolução. Para a avaliação do estado nutricional energético-proteico, foram utilizados peso e estatura pelos índices: peso/idade, estatura/idade e peso/estatura em escores-Z. As proporções foram comparadas pelo teste do Qui-quadrado de Pearson e teste exato de Fisher, e a associação entre as concentrações de hemoglobina e de retinolemia e o estado nutricional, pelo modelo de regressão de Poisson. RESULTADOS: Observou-se prevalência de 36,5% (IC95%=33,7-39,3) de anemia (Hb<11,0 g/dL), 21,4% (IC95%=17,3-22,2) de hipovitaminose A (<0,70µmol/L), 7,3% (IC95%=5,8-8,8)) de desnutrição crônica (estatura/idade<-2), 1,3% (IC95%=0,6-2,0) de desnutrição global (peso/idade<-2), e 2,3% (IC95%=1,4-3,2) de desnutrição aguda (estatura/idade<-2). Na análise multivariada, observou-se associação direta entre anemia e retinolemia inadequada, assim como entre anemia e desnutrição crônica. CONCLUSÃO: A elevada prevalência da anemia e da hipovitaminose A impõe a adoção de medidas efetivas de prevenção e controle. A associação entre as carências nutricionais demonstrou que uma melhoria na retinolemia bem como no estado nutricional tende a reduzir a anemia no contexto estudado