The pollen extracellular matrix contains proteins mediating species specificity and components needed for efficient pollination. We identified all proteins >10 kilodaltons in the Arabidopsis pollen coating and showed that most of the corresponding genes reside in two genomic clusters. One cluster encodes six lipases, whereas the other contains six lipid-binding oleosin genes, including GRP17, a gene that promotes efficient pollination. Individual oleosins exhibit extensive divergence between ecotypes, but the entire cluster remains intact. Analysis of the syntenic region in Brassica oleracea revealed even greater divergence, but a similar clustering of the genes. Such allelic flexibility may promote speciation in plants.

Flavin-binding LOV domains are blue-light photosensory modules that are conserved in a number of developmental and circadian regulatory proteins in plants, algae, and fungi. LOV domains are also present in bacterial genomes, and are commonly located at the amino termini of sensor histidine kinases. Genes predicted to encode LOV-histidine kinases are conserved across a broad range of bacterial taxa, from aquatic oligotrophs to plant and mammalian pathogens. However, the function of these putative prokaryotic photoreceptors remains largely undefined. The differentiating bacterium, Caulobacter crescentus, contains an operon encoding a two-component signaling system consisting of a LOV-histidine kinase, LovK, and a single-domain response regulator, LovR. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays increased ATPase and autophosphorylation activity in response to visible light. Deletion of the response regulator gene, lovR, results in severe attenuation of cell attachment to a glass surface under laminar flow, whereas coordinate, low-level overexpression of lovK and lovR results in a light-independent increase in cell-cell attachment, a response that requires both the conserved histidine phosphorylation site in LovK and aspartate phosphorylation site in LovR. Growing C. crescentus in the presence of blue light dramatically enhances cell-cell attachment in the lovKlovR overexpression background. A conserved cysteine residue in the LOV domain of LovK, which forms a covalent adduct with the flavin cofactor upon absorption of visible light, is necessary for the light-dependent regulation of LovK enzyme activity and is required for the light-dependent enhancement of intercellular attachment.Caulobacter ͉ LOV domain ͉ photoreceptor ͉ signal transduction ͉ histidine kinase P roteins that serve as detectors of environmental signals are often modular, containing conserved sensory domains that control diverse signaling outputs (1, 2). One such sensory module is the PAS (Per-ARNT-Sim) domain, which is conserved across all kingdoms of life and is capable of specifically binding a wide range of ligands including heme, flavins, p-coumaric acid, citrate, and other small molecules (3). A subclass of PAS domains, known as LOV domains for their role as sensors of light, oxygen, or voltage, commonly bind a flavin cofactor and function to regulate a number of blue light-dependent processes in plants and fungi (4). These photosensory LOV domains signal by means of a unique photocycle in which photon absorption drives the reversible formation of a covalent adduct between the 4a carbon of the flavin isoalloxazine ring and a conserved cysteine residue (5, 6). Adduct formation is followed by a large structural change at the C terminus of the LOV domain that leads to cell signaling (7,8). Beyond plants and fungi, dozens of proteins containing LOV photosensory domains have been identified in bacterial species (4, 9). Examples of bacterial LOV photosensors include LOV-phosphodiesterases, LOV-HTH transcription fa...

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition , we have identified a fertile, intragenic suppressor, cer6-2R , that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids Ͼ 28 carbons long. DNA sequence analysis revealed the nature of the cer6-1 , cer6-2 , and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1 , a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60 , with a high degree of nucleotide and amino acid similarity to CER6 , resides at the original CUT1 locus.

In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA) that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ). Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a ‘nutritional override’ system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.

A conserved set of regulators control the general stress response in Caulobacter crescentus , including σ T , its anti-σ factor NepR, the anti-anti-σ factor PhyR, and the transmembrane sensor kinase PhyK. We report that the soluble histidine kinase LovK and the single-domain response regulator LovR also function within the C. crescentus general stress pathway. Our genetic data support a model in which LovK-LovR functions upstream of σ T by controlling the phosphorylation state and thus anti-anti-σ activity of PhyR. Transcription of lovK and lovR is independently activated by stress through a mechanism that requires sigT and phyR . Conversely, lovK and lovR function together to repress transcription of the general stress regulon. Concordant with a functional role of the LovK-LovR two-component system as a negative regulator of the general stress pathway, lovK - lovR -null mutants exhibit increased cell survival after osmotic stress, while coordinate overexpression of lovK and lovR attenuates cell survival relative to that of the wild type. Notably, lovK can complement the transcriptional and cell survival defects of a phyK -null mutant when lovR is deleted. Moreover, in this same genetic background, σ T -dependent transcription is activated in response to osmotic stress. This result suggests that flavin-binding LOV (light, oxygen, or voltage) histidine kinases are competent to perceive cytoplasmic signals in addition to the environmental signal blue light. We conclude that the PhyK-PhyR and LovK-LovR two-component signaling systems coordinately regulate stress physiology in C. crescentus .

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.

No abstract

SummaryPhyR is a hybrid stress regulator conserved in a-proteobacteria that contains an N-terminal s-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-s factor. PhyR thus functions as an anti-anti-s factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as s T and its anti-s factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti-anti-s regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions s2 and s4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of a4-b5-a5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions s 2 and s4 in the SL domain to open about a flexible connector loop and bind anti-s factor.