The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by onblot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An overrepresentation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

SummaryArabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded L-isoaspartyl residues, arising spontaneously at L-asparaginyl and L-aspartyl sites in proteins, to L-aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5¢-and 3¢-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting L-isoaspartate to L-aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome.

The members of early auxin response gene family, Aux/IAA, encode negative regulators of auxin signaling but play a central role in auxin‐mediated plant development. Here we report the interaction of an Aux/IAA protein, AtIAA14, with Drought‐induced‐19 (Di19‐3) protein and its possible role in auxin signaling. The Atdi19‐3 mutant seedlings develop short hypocotyl, both in light and dark, and are compromised in temperature‐induced hypocotyl elongation. The mutant plants accumulate more IAA and also show altered expression of NIT2, ILL5, and YUCCA genes involved in auxin biosynthesis and homeostasis, along with many auxin responsive genes like AUX1 and MYB77. Atdi19‐3 seedlings show enhanced root growth inhibition when grown in the medium supplemented with auxin. Nevertheless, number of lateral roots is low in Atdi19‐3 seedlings grown on the basal medium. We have shown that AtIAA14 physically interacts with AtDi19‐3 in yeast two‐hybrid (Y2H), bimolecular fluorescence complementation, and in vitro pull‐down assays. However, the auxin‐induced degradation of AtIAA14 in the Atdi19‐3 seedlings was delayed. By expressing pIAA14::mIAA14‐GFP in Atdi19‐3 mutant background, it became apparent that both Di19‐3 and AtIAA14 work in the same pathway and influence lateral root development in Arabidopsis. Gain‐of‐function slr‐1/iaa14 (slr) mutant, like Atdi19‐3, showed tolerance to abiotic stress in seed germination and cotyledon greening assays. The Atdi19‐3 seedlings showed enhanced sensitivity to ethylene in triple response assay and AgNO3, an ethylene inhibitor, caused profuse lateral root formation in the mutant seedlings. These observations suggest that AtDi19‐3 interacting with AtIAA14, in all probability, serves as a positive regulator of auxin signaling and also plays a role in some ethylene‐mediated responses in Arabidopsis.Significance StatementThis study has demonstrated interaction of auxin responsive Aux/IAA with Drought‐induced 19 (Di19) protein and its possible implication in abiotic stress response.

F‐box (FB) proteins that form part of SKP1‐CUL1‐F‐box (SCF) type of E3 ubiquitin ligases are important components of plant growth and development. Here we characterized OsFBX257, a rice FB protein‐coding gene that is differentially expressed under drought conditions and other abiotic stresses. Population genomics analysis suggest that OsFBX257 shows high allelic diversity in aus accessions and has been under positive selection in some japonica, aromatic and indica cultivars. Interestingly, allelic variation at OsFBX257 in aus cultivar Nagina22 is associated with an alternatively spliced transcript. Conserved among land plants, OsFBX257 is a component of the SCF complex, can form homomers and interact molecularly with the 14‐3‐3 rice proteins GF14b and GF14c. OsFBX257 is co‐expressed in a network involving protein kinases and phosphatases. We show that OsFBX257 can bind the kinases OsCDPK1 and OsSAPK2, and that its phosphorylation can be reversed by phosphatase OsPP2C08. OsFBX257 expression level modulates root architecture and drought stress tolerance in rice. OsFBX257 knockdown (OsFBX257KD) lines show reduced total root length and depth, crown root number, panicle size and survival under stress. In contrast, its overexpression (OsFBX257OE) increases root depth, leaf and grain length, number of panicles, and grain yield in rice. OsFBX257 is a promising breeding target for alleviating drought stress‐induced damage in rice.