In flowering plants, the interaction of pollen tubes with female tissues is important for the accomplishment of double fertilization. Little information is known about the mechanisms that underlie signalling between pollen tubes and female tissues. In this study, two Arabidopsis pollen tube-expressed CrRLK1L protein kinases, Buddha's Paper Seal 1 (BUPS1) and BUPS2, were identified as being required for normal tip growth of pollen tubes in the pistil. They are expressed prolifically in pollen and pollen tubes and are localized on the plasma membrane of the pollen tube tip region. Mutations in BUPS1 drastically reduced seed set. Most of the bups1 mutant pollen tubes growing in the pistil exhibited a swollen pollen tube tip, leading to failure of fertilization. The bups2 pollen tubes had a slightly abnormal morphology but could still accomplish double fertilization. The bups1 bups2 double mutant exhibited a slightly enhanced phenotype compared to the single bups1 mutants. The BUPS1 proteins could form homomers and heteromers with BUPS2, whereas BUPS2 could only form heteromers with BUPS1. The BUPS proteins could interact with the Arabidopsis pollen-expressed RopGEFs in the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. The results indicated that the BUPSs may mediate normal polar growth of pollen tubes in the pistil.

The activation of aquaporins by a receptor kinase complex of SIRK1 and QSK1 was studied in detail. Based on phosphoproteomics, pulldown studies and physiological experiments we conclude that SIRK1 may function as a main receptor which forms a complex with coreceptor QSK1. SIRK1 can autophosphorylate and then trans-phosphorylate QSK1. Phosphorylated QSK1 enhanced and stabilized the interaction with aquaporins as substrates of the receptor kinase complex.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

These authors contributed equally to this work. SUMMARYIn flowering plants, male gametes (sperm cells) develop within male gametophytes (pollen grains) and are delivered to female gametes for double fertilization by pollen tubes. Therefore, pollen tube growth is crucial for reproduction. The mechanisms that control pollen tube growth remain poorly understood. In this study, we demonstrated that the ARID-HMG DNA-binding protein AtHMGB15 plays an important role in pollen tube growth. This protein is preferentially expressed in pollen grains and pollen tubes and is localized in the vegetative nuclei of the tricellular pollen grains and pollen tubes. Knocking down AtHMGB15 expression via a Ds insertion caused retarded pollen tube growth, leading to a significant reduction in the seed set. The athmgb15-1 mutation affected the expression of 1686 genes in mature pollen, including those involved in cell wall formation and modification, cell signaling and cellular transport during pollen tube growth. In addition, it was observed that AtHMGB15 binds to DNA in vitro and interacts with the transcription factors AGL66 and AGL104, which are required for pollen maturation and pollen tube growth. These results suggest that AtHMGB15 functions in pollen tube growth through the regulation of gene expression.

Mass spectrometry (MS)-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed “ShortPhos,” an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. “ShortPhos” can produce label-free datasets with a high quantitative reproducibility. In addition, the “ShortPhos” protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied “ShortPhos” to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The “ShortPhos” identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.