PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses.

Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

Bioinformatic Identification and Analysis of Hydroxyproline-Rich Glycoproteins in Populus trichocarpa.

BACKGROUND: Hydroxyproline-rich glycoproteins (HRGPs) constitute a plant cell wall protein superfamily that functions in diverse aspects of growth and development. This superfamily contains three members: the highly glycosylated arabinogalactan-proteins (AGPs), the moderately glycosylated extensins (EXTs), and the lightly glycosylated proline-rich proteins (PRPs). Chimeric and hybrid HRGPs, however, also exist. A bioinformatics approach is employed here to identify and classify AGPs, EXTs, PRPs, chimeric HRGPs, and hybrid HRGPs from the proteins predicted by the completed genome sequence of poplar (Populus trichocarpa). This bioinformatics approach is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs with a newly revised and improved BIO OHIO 2.0 program. Proteins detected by the program are subsequently analyzed to identify the following: 1) repeating amino acid sequences, 2) signal peptide sequences, 3) glycosylphosphatidylinositol lipid anchor addition sequences, and 4) similar HRGPs using the Basic Local Alignment Search Tool (BLAST). RESULTS: The program was used to identify and classify 271 HRGPs from poplar including 162 AGPs, 60 EXTs, and 49 PRPs, which are each divided into various classes. This is in contrast to a previous analysis of the Arabidopsis proteome which identified 162 HRGPs consisting of 85 AGPs, 59 EXTs, and 18 PRPs. Poplar was observed to have fewer classical EXTs, to have more fasciclin-like AGPs, plastocyanin AGPs and AG peptides, and to contain a novel class of PRPs referred to as the proline-rich peptides. CONCLUSIONS: The newly revised and improved BIO OHIO 2.0 bioinformatics program was used to identify and classify the inventory of HRGPs in poplar in order to facilitate and guide basic and applied research on plant cell walls. The newly identified poplar HRGPs can now be examined to determine their respective structural and functional roles, including their possible applications in the areas plant biofuel and natural products for medicinal or industrial uses. Additionally, other plants whose genomes are sequenced can now be examined in a similar way using this bioinformatics program which will provide insight to the evolution of the HRGP family in the plant kingdom.

3-Aminobenzamide Blocks MAMP-Induced Callose Deposition Independently of Its Poly(ADPribosyl)ation Inhibiting Activity.

Cell wall reinforcement with callose is a frequent plant response to infection. Poly(ADP-ribosyl)ation is a protein post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs). Poly(ADP-ribosyl)ation has well-known roles in DNA damage repair and has more recently been shown to contribute to plant immune responses. 3-aminobenzamide (3AB) is an established PARP inhibitor and it blocks the callose deposition elicited by flg22 or elf18, two microbe-associated molecular patterns (MAMPs). However, we report that an Arabidopsis parp1parp2parp3 triple mutant does not exhibit loss of flg22-induced callose deposition. Additionally, the more specific PARP inhibitors PJ-34 and INH2BP inhibit PARP activity in Arabidopsis but do not block MAMP-induced callose deposition. These data demonstrate off-target activity of 3AB and indicate that 3AB inhibits callose deposition through a mechanism other than poly(ADP-ribosyl)ation. POWDERY MILDEW RESISTANT 4 (PMR4) is the callose synthase responsible for the majority of MAMP- and wound-induced callose deposition in Arabidopsis. 3AB does not block wound-induced callose deposition, and 3AB does not reduce the PMR4 mRNA abundance increase in response to flg22. Levels of PMR4-HA protein increase in response to flg22, and increase even more in flg22 + 3AB despite no callose being produced. The callose synthase inhibitor 2-deoxy-D-glucose does not cause similar impacts on PMR4-HA protein levels. Beyond MAMPs, we find that 3AB also reduces callose deposition induced by powdery mildew (Golovinomyces cichoracearum) and impairs the penetration resistance of a PMR4 overexpression line. 3AB thus reveals pathogenesis-associated pathways that activate callose synthase enzymatic activity distinct from those that elevate PMR4 mRNA and protein abundance.

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.

IRX14 and IRX14-LIKE, two glycosyl transferases involved in glucuronoxylan biosynthesis and drought tolerance in Arabidopsis.

IRX14 and IRX14-LIKE (IRX14L) are two closely related glycosyl transferases in the glycosyl transferase 43 (GT43) family of Arabidopsis. A T-DNA insertion mutant for IRX14 results in comparatively minor changes, such as irregular xylem, while a mutation for IRX14L results in no changes. However, an irx14 and irx14L double mutant severely affects growth and development, with the dwarf plants failing to produce an inflorescence stem. Plants that are homozygous for IRX14 but heterozygous for IRX14L (irx14 irx14L(±)) exhibit an intermediate phenotype, including noticeably smaller leaves, stems, and underdeveloped siliques. Additionally, the T-DNA insertion mutant for IRX14 was found to result in a drought-tolerant phenotype. Carbohydrate analysis of total cell wall extracts revealed a reduction in xylose for the irx14 and irx14 irx14L(±) mutants, consistent with a defect in glucuronoxylan biosynthesis. Immunolocalization of xylan with the LM10 antibody revealed a loss of xylan in irx14 mutants and a further reduction in the irx14 irx14L(±) mutants. IRX14L likely functions redundantly with IRX14 in glucuronoxylan biosynthesis, with IRX14 having a more important role in the process.