Transcriptional and Metabolic Adjustments in ADP-Glucose Pyrophosphorylase-Deficientbt2Maize Kernels  

Abstract

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 1H 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).

Functions of Multiple Genes Encoding ADP-Glucose Pyrophosphorylase Subunits in Maize Endosperm, Embryo, and Leaf

“…The RT-PCR data collected here (Fig. 1B), together with previously published RNA gel-blot and RT-PCR analyses of some of these gene/tissue combinations Rösti and Denyer, 2007;Cossegal et al, 2008;Yan et al, 2009;Huang et al, 2010), agree closely with the RNASeq results. AGPLLZM, AGPLEMZM, and SH2 mRNAs encoding AGPase LS and AGPSLZM, AGPSEMZM, BT2a, and BT2b transcripts encoding AGPase SS were all detected in endosperm by RT-PCR.…”

“…Three such transcripts encoding AGPase SS, namely BT2b, AGPSLZM, and AGPSEMZM, have been detected in maize endosperm and could specify plastidial proteins Rösti and Denyer, 2007;Cossegal et al, 2008). Two transcripts other than SH2 that encode AGPase LS, namely AGPLEMZM (formerly referred to as agp1; Giroux and Hannah, 1994;Giroux et al, 1995) and AGPLLZM (for AGPase large subunit from a leaf cDNA of Zea mays; previously referred to as Zmagpl1 or AGPL4; Yan et al, 2009;Huang et al, 2010), are present in maize endosperm.…”

“…A complementary DNA (cDNA) from a type 2 gene encoding AGPase SS, termed agpsemzm (formerly referred to as agp2), was cloned from embryo tissue Giroux et al, 1995). In addition, a third gene encoding AGPase SS, termed agpslzm (formerly referred to as L2), is present in maize owing to a duplication of the ancestral type 1 gene (Prioul et al, 1994;Hannah et al, 2001;Rösti and Denyer, 2007;Cossegal et al, 2008). Maize differs from other cereals in which type 1a and 1b transcripts from the same gene specify AGPase SS in endosperm cytosol and leaf plastids, respectively.…”

Megabase Level Sequencing Reveals Contrasted Organization and Evolution Patterns of the Wheat Gene and Transposable Element Spaces

“…The spotted membranes were incubated at 378C on Luria-Bertani agar with 12.5 mg/mL chloramphenicol and were treated as described by Paux et al (2004). Total RNAs of hexaploid wheat cv Chinese Spring were extracted from 500 mg of five organs (root, leaf, stem, spike, and grain) at two or three developmental stages each as described by Cossegal et al (2008). DNA was removed using RQ1 RNase-free DNase (Promega).…”

ADP-Glucose Pyrophosphorylase-Deficient Pea Embryos Reveal Specific Transcriptional and Metabolic Changes of Carbon-Nitrogen Metabolism and Stress Responses

“…Its overexpression in Vicia narbonensis seeds channels more C into organic acids, stimulates amino acid biosynthesis, and increases protein content (Rolletschek et al, 2004;Radchuk et al, 2007). Accumulation of Suc, as shown for AGP-repressed Vicia (Weber et al, 2000;Rolletschek et al, 2002) and maize (Cossegal et al, 2008) seeds, causes repartitioning of C into glycolysis, tricarboxylic acid (TCA) cycle, and amino acid biosynthesis.…”