RESUMEN

During the cloning of monogenic recessive mutations responsible for a defective kernel phenotype in a Mutator-induced Zea mays mutant collection, we isolated a new mutant allele in Brittle2 (Bt2), which codes for the small subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch synthesis. Reverse transcription-polymerase chain reaction experiments with gene-specific primers confirmed a predominant expression of Bt2 in endosperm, of Agpsemzm in embryo, and of Agpslzm in leaf, but also revealed considerable additional expression in various tissues for all three genes. Bt2a, the classical transcript coding for a cytoplasmic isoform, was almost exclusively expressed in the developing endosperm, whereas Bt2b, an alternative transcript coding for a plastidial isoform, was expressed in almost all tissues tested with a pattern very similar to that of Agpslzm. The phenotypic analysis showed that, at 30 d after pollination (DAP), mutant kernels were plumper than wild-type kernels, that the onset of kernel collapse took place between 31 and 35 DAP, and that the number of starch grains was greatly reduced in the mutant endosperm but not the mutant embryo. A comparative transcriptome analysis of wild-type and bt2-H2328 kernels at middevelopment (35 DAP) with the 18K GeneChip Maize Genome Array led to the conclusion that the lack of Bt2-encoded AGPase triggers large-scale changes on the transcriptional level that concern mainly genes involved in carbohydrate or amino acid metabolic pathways. Principal component analysis of (1)H nuclear magnetic resonance metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not only affect the endosperm, but also the embryo at the metabolic level. These data suggest that, in the bt2-H2328 endosperms, regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo).

Asunto(s)

Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Transcripción Genética , Zea mays/metabolismo , Secuencia de Bases , Cartilla de ADN , Perfilación de la Expresión Génica , Genes de Plantas , Glucosa-1-Fosfato Adenililtransferasa/genética , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , ARN Mensajero/genética , Zea mays/enzimología , Zea mays/genética

RESUMEN

• The cellular tolerance to nickel (Ni), zinc (Zn) and cadmium (Cd) of two poly-hyperaccumulators, Arabidopsis halleri and Thlaspi caerulescens, was investigated in order to compare their cellular phenotypes toward various metal ion exposures. • Protoplasts were kept for 24 h on solutions containing increasing concentrations of the metal ions, and a viability test was performed. Zinc loading of the protoplasts was investigated with Arabidopsis lyrata and A. halleri protoplasts using the Zn fluorescent indicator Newport green diacetate. • Only T. caerulescens protoplasts showed a clear tolerance to Ni. On the other hand, protoplasts from both hyperaccumulators displayed a very high and constitutive Zn tolerance and an inducible Cd tolerance. The vacuolar storage of Zn was confirmed, but no Zn accumulation at all was observed in A. halleri protoplasts after Zn exposure. • Specific metal tolerances were found at the cellular level in the hyperaccumulating plants, highlighting that specific adaptations to metal ions exist in the cells as well as in the whole plants.