DOI: 10.25338/b8bs75
title: Nectar bacteria stimulate pollen germination and bursting to enhance microbial fitness

description: Many organisms consume pollen, yet mechanisms of its digestion remains a
 fundamental enigma in pollination biology, as pollen is protected by a
 recalcitrant outer shell. Pollen is commonly found in floral nectar, as
 are nectar microbes, which are nearly ubiquitous among flowers. Nectar
 specialist bacteria, like Acinetobacter, can reach high densities (up to
 109 cells/mL), despite the fact that floral nectar is nitrogen poor. Here,
 we show evidence that the genus Acinetobacter, a prevalent nectar- and
 bee-associated bacteria, can induce pollen germination and bursting, gain
 access to protoplasm nutrients, and thereby grow to higher densities.
 Although induced germination has been suggested as a potential method in
 macroscopic pollen consumers, and fungal inhibition of pollen germination
 has been shown, direct biological induction of germination has not been
 empirically documented outside of plants. Acinetobacter pollinis SCC477
 induced over 5x greater pollen germination and 20x greater pollen bursting
 than that of uninoculated pollen by 45 minutes. When provided with
 germinable pollen, A. pollinis stimulates protein release and grows to
 nearly twice the density compared to growth with ungerminable pollen,
 indicating that stimulation of germination benefits bacterial fitness. In
 contrast, a common nectar-inhabiting yeast did not affect pollen
 germination nor benefit from it. We conclude that Acinetobacter both
 specifically causes and benefits from inducing pollen germination and
 bursting. Further study of microbe-pollen interactions may inform many
 aspects of pollination ecology, including floral microbial ecology,
 pollinator nutrient acquisition from pollen, and cues of pollen
 germination for plant reproduction.
description: <strong>**FILES' NAMES AND
 CONTENTS**</strong><br> pollen_microbe_data.csv<br> -
 The file is a comma delimited file. Data on pollen germination and
 bursting with different microbial treatments
 MALDI_identifications.csv<br> - The file is a comma
 delimited file. Data on identifications of microbes after assay completion
 using MALDI-TOF + Compass Explorer
 dose_dependence-data.csv<br> - The file is a comma
 delimited file. Data on proportion of grains germinated with different
 dilutions of A. pollinisSCC477 over time.
 tube_lengths_dose_dependence_data.csv<br> - The file is a
 comma delimited file. Data on lengths of pollen tubes over time and with
 different dilutions of A. pollinisSCC477
 growthcurver_data_dose_dep_each_well.csv<br> - The file is
 a comma delimited file. Data is formatted from
 'dose_dependence-data.csv' to work with "GrowthCurver"
 package.
 growthcurver_data_dose_dep_by_treatment.csv<br> - The file
 is a comma delimited file. Data is from
 'growthcurver_dose_dep_each_well.csv' as averages of the three
 replicates, and in the format for use by  "GrowthCurver"
 package. Apollinis_benefit_data.csv<br> - The
 file is a comma delimited file. Data on microbial hemocytometer counts of
 A. pollinis, when provided different pollen treatments. 
 yeast_benefit_data.csv<br> - The file is a comma delimited
 file. Data on microbial hemocytometer counts of M. reukaufii, when
 provided different pollen treatments.
 Lowry_data1.csv<br> - The file is a comma delimited file.
 Data on protein in solution after pollen (germinable or ungerminable or no
 pollen) exposure to SCC477 (or no inoculation). 
 germ_AN.csv<br> - The file is a comma delimited file. Data
 on germination and bursting after pollen (germinable or ungerminable)
 exposure to SCC477 (or no inoculation).  <br>
 =================================================================================== **FILES' STRUCTURES AND UNITS OF MEASUREMENTS** pollen_microbe_data.csv<br> The file is a comma delimited file.<br> Column headings are in the following order and format: Microbe.group    <br> - The classification of the microbe added  Microbetreatment<br> - The integrally used strain number of the microbe added (coded to species name in R code)<br>     <br> Trial.date<br> - The date of the assay<br>     <br> Assay.replicate<br> - The biological replicate (within microbe treatment)  Timepoint<br> - In minutes, the time elapsed since microbes and pollen were mixed in the assay<br>     <br> standardized.OD.plate<br> - The OD600 of the microbe when mixed in the plate (so diluted 1:1 from initial OD600s of .1 and .05) Total.pollen    <br> - The total number of pollen grains captured in the image, not including those that touch the outer perimeter of the image<br>  <br> number.germ<br> - The number of pollen grains with protruding pollen tubes in the image that are intact (not "tip burst")      number.burst    <br> - The number of pollen grains with no visible pollen tube, but releasing protoplasm number.tip.burst<br> - The number of grains that germinated and the tip of the tube released protoplasm<br>     <br> Prop.germ<br> - The proportion of pollen grains that germinated (eg number.germ/Total.pollen)<br>     <br> Prop.burst<br> - The proportion of pollen grains that burst (eg number.burst/Total.pollen)<br>     <br> Prop.tip.burst    <br> - The proportion of pollen grains that burst at the tip of the pollen tube (eg. number.tip.burst/ Total.pollen) allgerm    <br> - The proportion of pollen grains that germinated, including both "number.germ" and "number.tip.burst"   allburst<br> - The proportion of pollen grains that expelled protoplasm into solution, including both "number.burst" and "number.tip.burst"  ===================================================================================<br> MALDI_identifications.csv The file is a comma delimited file. Column headings are in the following order and format: Trial Date<br> - The date of the assay for which the identifications are being made     Inoculation source:<br> - The well of the assay plate that the microbe was plated (onto agar plate) from.  Microbe Inoculated:    <br> - The microbe that was added, or if none, what the source of the microbes were (eg pollen plated directly without inoculation)  Microbe IDed on MALDI:    <br> - The microbe that the MALDI-TOF + Compass Explorer identified as the top hit for that spot NCBI identifier    Score    <br> - The NCBI identifier of the microbe that Compass Explorer identified as the top hit for that spot Genus match?    <br> - "yes" if genus IDed was the same as genus inoculated Species match?    <br> - "yes" if species IDed was the same as species inoculated Trial Type<br> - "Dose Dependence" for the trial of A. pollinis dose comparison, "Microbe Assay" for the trials comparing different microbes for their effects on pollen notes<br> - important caveats and information that did not fit elsewhere<br> ===================================================================================<br> dose_dependence_data.csv  The file is a comma delimited file.  Column headings are in the following order and format: Trial date    <br> - The date of the assay image ID    <br> - The ID of the image used, corresponds to the dilution (letter) and replicate (number) Microbetreatment<br> - The dilution of A. pollinis used <br>     <br> Assay replicate    <br> - The biological replicate, 3 per dilution level Minutes    <br> - Time in minutes since microbes and pollen were mixed in the assay Total.pollen    <br> - The total number of pollen grains captured in the image, not including those what touch the outer perimeter of the image<br>  <br> number.germ<br> - The number of pollen grains with protruding pollen tubes in the image that are intact (not "tip burst")      number.burst    <br> - The number of pollen grains with no visible pollen tube, but releasing protoplasm number.tip.burst<br> - The number of grains that germinated and the tip of the tube released protoplasm<br>     <br> Prop.germ<br> - The proportion of pollen grains that germinated (eg number.germ/Total.pollen)<br>     <br> Prop.burst<br> - The proportion of pollen grains that burst (eg number.burst/Total.pollen)<br>     <br> Prop.tip.burst    <br> - The proportion of pollen grains that burst at the tip of the pollen tube (eg. number.tip.burst/ Total.pollen) allgerm    <br> - The proportion of pollen grains that germinated, including both "number.germ" and "number.tip.burst"   allburst<br> - The proportion of pollen grains that expelled protoplasm into solution, including both "number.burst" and "number.tip.burst"  ===================================================================================<br> tube_lengths_dose_dependence_data.csv The file is a comma delimited file. Data is structured in columns, each row representing a single pollen tube. Column headings are in the following order and format: Minutes<br> - The number minutes elapsed since T0 Time_l<br> - The time label, in minutes (m) or hours (h) since T0 Dilution<br> - The dilution of A. pollinisSCC477 used in that well Well<br> - The ID of the well (letter corresponds to dilution, number to replicate)  Tube number<br> - Differentiates tubes within a well length<br> - The length of the germinated tube, in (um), when traced along the center line of the tube.  <br> ===================================================================================<br> growthcurver_data_dose_dep_each_well.csv  The file is a comma delimited file. Data is structured with 'time' as the first column, and individual wells (replicates) as other columns. This is the required format for the growcurver package used in the dose dependence curve analysis R script time <br> - In minutes. The other columns are named with the letter encoding the dilution, and the number indicating the replicate (3 replicates of each) <br>     a= full dose (OD .1 A. pollinis)<br>     b= 1:2 dilution of OD .1 A. pollinis<br>     c= 1:3<br>     d= 1:5<br>     e= 1:20<br>     f= 1:100<br>     g= no microbes added <br> ===================================================================================<br> growthcurver_data_dose_dep_by_treatment.csv  The file is a comma delimited file. Data is structured with 'time' as the first column, and averages of the three individual wells (replicates) in the other columns. This is the required format for the growcurver package used in the dose dependence curve analysis R script Column headings are in the following order and format time    <br> - In minutes full_dose<br> - The proportion of germinated grains at each timepoint when A. pollinis solution added was the full dose (OD600= 0.1) dil2<br> - The proportion of germinated grains at each timepoint when A. pollinis diluted 1:2<br>     <br> dil3    <br> - The proportion of germinated grains at each timepoint when A. pollinis diluted 1:3 dil5    <br> - The proportion of germinated grains at each timepoint when A. pollinis diluted 1:5 dil20    <br> - The proportion of germinated grains at each timepoint when A. pollinis diluted 1:20 dil100    <br> - The proportion of germinated grains at each timepoint when A. pollinis diluted 1:100 no_microbes<br> - The proportion of germinated grains at each timepoint when sterile BK added, no microbes <br> ===================================================================================<br> Apollinis_benefit_data.csv<br> - The file is a comma delimited file. Column headings are in the following order and format: Treatment_l<br> - Indicates the treatment of the pollen and whether or not A. pollinis was added. FP= Fresh pollen (directly from flowers) UP= ungerminable pollen (microwaved for 3 minutes in liquid BK media to render unable to germinate)  Tube<br> - The tube label, three replicates per treatment initial count<br> - The number of cells per mL at the start of the assay for each replicate, as counted on hemocytometer  final count-24h <br> - The number of cells per mL at the 24 hour timepoint for each replicate, as counted on hemocytometer Date<br> - The date the assay was started (day of initial count) =================================================================================== yeast_benefit_data.csv<br> - The file is a comma delimited file. Data on microbial hemocytometer counts of M. reukaufii, when provided different pollen treatments.  <br> Column headings are in the following order and format: Treatment_l<br> - Indicates the treatment of the pollen and whether or not M. reukauffi was added. FP= Fresh pollen (directly from flowers) UP= ungerminable pollen (microwaved for 3 minutes in liquid BK media to render unable to germinate)  initial count<br> - The number of cells per mL at the start of the assay for each replicate, as counted on hemocytometer  24h count<br> - The number of cells per mL at the 24 hour timepoint for each replicate, as counted on hemocytometer Date<br> - The date the assay was started (day of initial count)<br> =================================================================================== Lowry_data1.csv<br> - The file is a comma delimited file. Data on protein in solution after pollen (germinable or ungerminable or no pollen) exposure to SCC477 (or no inoculation).  Column headings are in the following order and format: row    <br> - Indicates the row of the 96 well plate column    <br> - Indicates the column of the 96 well plate  Timepoint<br> - The time at which the measurement was taken relative to when microbes and pollen were combined in the plate.     Well ID     - The combined column and row values<br>     <br> protein <br> - The ug/mL of proetein In the well after correction by dilution factor (4) Treatment<br> - Combined pollen and microbe treatment microbetreatment<br> - Indicates presence (477) or absence (ctrl) of SCC477 pollentreatment<br> - Indicates pollen treatment (fresh= germinable pollen,  ungerm= ungerminable, or no pollen) Date<br> - Date the assay was started (eg time 0) =================================================================================== germ_AN.csv<br> - The file is a comma delimited file. Data on germination and bursting after pollen (germinable or ungerminable) exposure to SCC477 (or no inoculation).  Treatment<br> - Combined microbe and pollen treatment: 477= presence of SCC477, ctrl= no inoculation, GP= germinable pollen, UP= ungerminable pollen. pollentreatment    <br> - pollen treatment: GP= germinable pollen, UP= ungerminable pollen Timepoint<br> - The time at which the measurement was taken relative to when microbes and pollen were combined in the plate.     image name<br> - Image name corresponds to 96 well plate well location<br>     <br> total<br> - Total pollen grains in image     burst<br> - Number of burst grains in image<br>     <br> germ<br> - Number of germinated pollen grains in image<br>     <br> tipburst<br> - Number of pollen grains that have germinated the burst<br>     <br> allgerm    <br> - combined germinated and tip burst allburst<br> - combined tip burst and burst<br>     <br> Date<br> - Date the assay was started (eg time 0)      <strong>**CODE FOR FIGURES AND STATISTICS**</strong> F2_germ_burst_code.R - code for Figure 2 and associated statistics F3_pollen_benefit_code.R - code for Figure 3 and associated statistics F4_protein_release.R  - code for Figure 4 and associated statistics FS1_dose_dep_code.R - code for Figure S1 and associated statistics FS1_dose_dependence_tube_lengths_supplemental_code.R - code for Figure S1 D and associated statistics FS3_AN_germ_code.R - code for Figure S3 and associated statistics <strong>**VIDEO**</strong> Video_S1.mp4<br> - Example of pollen grains that germinated and how the tips of the tubes are bursting after 60m with A. pollinis_SCC477. It is a time-lapse video over 5 minutes. <br> - Example of pollen grains bursting after 60m with A. pollinis_SCC477. It is a time-lapse video over 8 minutes.  **DATA QUESTIONS**<br> Questions about data and code can be addressed to Shawn Christensen (smchristensen@ucdavis.edu)