The continuing rise in atmospheric CO2 causes closing of stomatal pores in leaves and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress1–4. However, the CO2-binding proteins that control this response remain unknown. Moreover, the cell type that responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate5–8. We demonstrate that Arabidopsis double mutant plants in the β-carbonic anhydrases, βCA1 and βCA4, display impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses.βCA-mediated CO2-triggered stomatal movements are not, in-first-order, linked to leaf-photosynthesis and can function in guard cells. Furthermore, guard cell βCA-over-expression plants exhibit enhanced water use efficiency. Guard cell-expression of mammalian αCAII complements ca1ca4 shows that carbonic anhydrase-mediated catalysis is an important mechanism for βCA-mediated CO2-induced stomatal closing and patch clamp analyses indicate that CO2/HCO3−transfers the signal to anion channel regulation. These findings, together with ht1-29 epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway that controls gas-exchange between plants and the atmosphere.

To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35STcDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 mM ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F 2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35STcDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35STAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABAhypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35STAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD 1 to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

Gene expression in plant mitochondria is still inadequately analyzed. To learn more about transcription and RNA processing in plant mitochondria, the 5'- and 3'-RNA extremities and the promoters of the cytochrome oxidase gene (cox2) were analyzed in pea. Both 5' and 3' ends of cox2 transcripts were examined by RT-PCR across the ligation site of circularized mitochondrial RNA as template. This approach identified 5' ends a few nucleotides shorter than three major 5' ends mapped by primer extension analysis. Presumably, only monophosphate 5' ends derived from processing can be ligated. In vitro transcription assays using a homologous mitochondrial protein extract from pea strongly suggest the major 5' ends to derive from transcription initiation. The cDNA analysis of the head-to-tail ligated cox2 mRNA identified 3' ends within a thymidine stretch approximately 300 nt downstream of the reading frame in a sequence segment that was not present in the previous investigation of this gene. Nuclease S1 protection experiments confirmed this newly identified 3' terminus and corroborated the validity of this technique in mRNA end analysis. The general use of the circularized RNA (CR)-RT-PCR approach for the simultaneous analysis of the 5' and 3' extremities of mRNA molecules is discussed.

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To determine the influence of posttranscriptional modifications on 3 end processing and RNA stability in plant mitochondria, pea atp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3 nonencoded nucleotides. A 3 rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3 ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3 poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3 nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3 processed and stable transcripts.Control of the amount of translatable mRNA is a common parameter to regulate gene expression in many organisms. The available quantity of an RNA depends largely on the rate of synthesis and/or posttranscriptional processes which control the stability of a transcript by preventing it from degradation. Such posttranscriptional processes have been studied in prokaryotes and different subcellular compartments of eukaryotic cells. In spite of the many variations, some common features behind these processes emerge in all organisms and systems. These include the involvement of mRNA secondary structures and 3Ј polyadenylation of RNA. While organized RNA secondary structures generally support transcript stabilization, polyadenylation has opposing functions in eukaryotes and prokaryotes.In the nucleus of eukaryotic cells, pre-mRNAs are polyadenylated at 3Ј ends that have been generated by endonucleolytic cleavages. These poly(A) tails play important roles in translation initiation and mRNA export from the nucleus and also have a major function in stabilizing mRNAs (27). A completely different function has been attributed to polyadenylation in prokaryotes, where the degradation of mRNA is stimulated by the presence of 3Ј poly(A) tracts (4). The degradation-promoting effect of polyadenylation was even found in transcripts, which were otherwise protected and stabilized by stem-loop structures (3). A similar effect has also been ob...

SummaryThe precise regulation of RNA metabolism has crucial roles in numerous developmental and physiological processes such as the induction of flowering in plants. Here we report the identification of processes associated with mRNA metabolism of flowering-time regulators in wild-type Arabidopsis plants, which were revealed by an early flowering mutation, abh1, in an Arabidopsis nuclear mRNA cap-binding protein. By using abh1 as an enhancer of mRNA metabolism events, we identify non-coding polyadenylated cis natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants. Our analyses also reveal a regulatory function of FLC intron 1 during transcript maturation in wild type. Moreover, transcripts encoding the FLM MADS box transcription factor are subject to premature intronic polyadenylation in wild type. In each case, abh1 showed altered patterns in RNA metabolism in these events compared with wild type. Together, abh1 enhances steps in the RNA metabolism that allowed us to identify novel molecular events of three key flowering-time regulators in wild-type plants, delivering important insights for further dissecting RNA-based mechanisms regulating flowering time in Arabidopsis.

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