A transcriptomics approach uncovers novel roles for poly(ADP-ribosyl)ation in the basal defense response in Arabidopsis thaliana.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) or loss of Arabidopsis thaliana PARG1 (poly(ADP-ribose) glycohydrolase) disrupt a subset of plant defenses. In the present study we examined the impact of altered poly(ADP-ribosyl)ation on early gene expression induced by the microbe-associate molecular patterns (MAMPs) flagellin (flg22) and EF-Tu (elf18). Stringent statistical analyses and filtering identified 178 genes having MAMP-induced mRNA abundance patterns that were altered by either PARP inhibitor 3-aminobenzamide (3AB) or PARG1 knockout. From the identified set of 178 genes, over fifty Arabidopsis T-DNA insertion lines were chosen and screened for altered basal defense responses. Subtle alterations in callose deposition and/or seedling growth in response to those MAMPs were observed in knockouts of At3g55630 (FPGS3, a cytosolic folylpolyglutamate synthetase), At5g15660 (containing an F-box domain), At1g47370 (a TIR-X (Toll-Interleukin Receptor domain)), and At5g64060 (a predicted pectin methylesterase inhibitor). Over-represented GO terms for the gene expression study included "innate immune response" for elf18/parg1, highlighting a subset of elf18-activated defense-associated genes whose expression is altered in parg1 plants. The study also allowed a tightly controlled comparison of early mRNA abundance responses to flg22 and elf18 in wild-type Arabidopsis, which revealed many differences. The PARP inhibitor 3-methoxybenzamide (3MB) was also used in the gene expression profiling, but pleiotropic impacts of this inhibitor were observed. This transcriptomics study revealed targets for further dissection of MAMP-induced plant immune responses, impacts of PARP inhibitors, and the molecular mechanisms by which poly(ADP-ribosyl)ation regulates plant responses to MAMPs.

Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids.

It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called "PABA synthases," although it is unclear whether they produce PABA or ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol.g(-1) fresh weight.

LeCTR1, a tomato CTR1-like gene, demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato.

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1 (4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1 and Arabidopsis CTR1. The introns in LeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsis ctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis.