We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase b-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase b-subunit in the dark.

This work investigated the roles of b-amylases in the breakdown of leaf starch. Of the nine b-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable b-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized b-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active b-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total b-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that b-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

A previously unknown maltose transporter is essential for the conversion of starch to sucrose in Arabidopsis leaves at night. The transporter was identified by isolating two allelic mutants with high starch levels and very high maltose, an intermediate of starch breakdown. The mutations affect a gene of previously unknown function, MEX1. We show that MEX1is a maltose transporter that is unrelated to other sugar transporters. The severe mex1 phenotype demonstrates that MEX1is the predominant route of carbohydrate export from chloroplasts at night. Homologous genes in plants including rice and potato indicate that maltose export is of widespread significance.

SummaryMaltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identi®ed in leaves of wild-type plants. Its activity was suf®cient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.