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Nectar bacteria stimulate pollen germination and bursting to enhance microbial fitness

Citation

Christensen, Shawn (2021), Nectar bacteria stimulate pollen germination and bursting to enhance microbial fitness , Dryad, Dataset, https://doi.org/10.25338/B8BS75

Abstract

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.

Usage Notes

**FILES' NAMES AND CONTENTS**
pollen_microbe_data.csv
- The file is a comma delimited file. Data on pollen germination and bursting with different microbial treatments

MALDI_identifications.csv
- The file is a comma delimited file. Data on identifications of microbes after assay completion using MALDI-TOF + Compass Explorer

dose_dependence-data.csv
- 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
- 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
- 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
- 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
- The file is a comma delimited file. Data on microbial hemocytometer counts of A. pollinis, when provided different pollen treatments. 

yeast_benefit_data.csv
- The file is a comma delimited file. Data on microbial hemocytometer counts of M. reukaufii, when provided different pollen treatments.

Lowry_data1.csv
- 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
- The file is a comma delimited file. Data on germination and bursting after pollen (germinable or ungerminable) exposure to SCC477 (or no inoculation). 


===================================================================================

**FILES' STRUCTURES AND UNITS OF MEASUREMENTS**

pollen_microbe_data.csv
The file is a comma delimited file.
Column headings are in the following order and format:

Microbe.group    
- The classification of the microbe added 

Microbetreatment
- The integrally used strain number of the microbe added (coded to species name in R code)
    
Trial.date
- The date of the assay
    
Assay.replicate
- The biological replicate (within microbe treatment) 

Timepoint
- In minutes, the time elapsed since microbes and pollen were mixed in the assay
    
standardized.OD.plate
- The OD600 of the microbe when mixed in the plate (so diluted 1:1 from initial OD600s of .1 and .05)

Total.pollen    
- The total number of pollen grains captured in the image, not including those that touch the outer perimeter of the image
 
number.germ
- The number of pollen grains with protruding pollen tubes in the image that are intact (not "tip burst")     

number.burst    
- The number of pollen grains with no visible pollen tube, but releasing protoplasm

number.tip.burst
- The number of grains that germinated and the tip of the tube released protoplasm
    
Prop.germ
- The proportion of pollen grains that germinated (eg number.germ/Total.pollen)
    
Prop.burst
- The proportion of pollen grains that burst (eg number.burst/Total.pollen)
    
Prop.tip.burst    
- The proportion of pollen grains that burst at the tip of the pollen tube (eg. number.tip.burst/ Total.pollen)

allgerm    
- The proportion of pollen grains that germinated, including both "number.germ" and "number.tip.burst"  

allburst
- The proportion of pollen grains that expelled protoplasm into solution, including both "number.burst" and "number.tip.burst" 

===================================================================================
MALDI_identifications.csv

The file is a comma delimited file.

Column headings are in the following order and format:

Trial Date
- The date of the assay for which the identifications are being made    

Inoculation source:
- The well of the assay plate that the microbe was plated (onto agar plate) from. 

Microbe Inoculated:    
- 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:    
- The microbe that the MALDI-TOF + Compass Explorer identified as the top hit for that spot

NCBI identifier    Score    
- The NCBI identifier of the microbe that Compass Explorer identified as the top hit for that spot

Genus match?    
- "yes" if genus IDed was the same as genus inoculated

Species match?    
- "yes" if species IDed was the same as species inoculated

Trial Type
- "Dose Dependence" for the trial of A. pollinis dose comparison, "Microbe Assay" for the trials comparing different microbes for their effects on pollen

notes
- important caveats and information that did not fit elsewhere
===================================================================================
dose_dependence_data.csv

 The file is a comma delimited file. 

Column headings are in the following order and format:

Trial date    
- The date of the assay

image ID    
- The ID of the image used, corresponds to the dilution (letter) and replicate (number)

Microbetreatment
- The dilution of A. pollinis used 
    
Assay replicate    
- The biological replicate, 3 per dilution level

Minutes    
- Time in minutes since microbes and pollen were mixed in the assay

Total.pollen    
- The total number of pollen grains captured in the image, not including those what touch the outer perimeter of the image
 
number.germ
- The number of pollen grains with protruding pollen tubes in the image that are intact (not "tip burst")     

number.burst    
- The number of pollen grains with no visible pollen tube, but releasing protoplasm

number.tip.burst
- The number of grains that germinated and the tip of the tube released protoplasm
    
Prop.germ
- The proportion of pollen grains that germinated (eg number.germ/Total.pollen)
    
Prop.burst
- The proportion of pollen grains that burst (eg number.burst/Total.pollen)
    
Prop.tip.burst    
- The proportion of pollen grains that burst at the tip of the pollen tube (eg. number.tip.burst/ Total.pollen)

allgerm    
- The proportion of pollen grains that germinated, including both "number.germ" and "number.tip.burst"  

allburst
- The proportion of pollen grains that expelled protoplasm into solution, including both "number.burst" and "number.tip.burst" 

===================================================================================
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
- The number minutes elapsed since T0

Time_l
- The time label, in minutes (m) or hours (h) since T0

Dilution
- The dilution of A. pollinisSCC477 used in that well

Well
- The ID of the well (letter corresponds to dilution, number to replicate) 

Tube number
- Differentiates tubes within a well

length
- The length of the germinated tube, in (um), when traced along the center line of the tube.  
===================================================================================
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 
- In minutes.

The other columns are named with the letter encoding the dilution, and the number indicating the replicate (3 replicates of each) 
    a= full dose (OD .1 A. pollinis)
    b= 1:2 dilution of OD .1 A. pollinis
    c= 1:3
    d= 1:5
    e= 1:20
    f= 1:100
    g= no microbes added


===================================================================================
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    
- In minutes

full_dose
- The proportion of germinated grains at each timepoint when A. pollinis solution added was the full dose (OD600= 0.1)

dil2
- The proportion of germinated grains at each timepoint when A. pollinis diluted 1:2
    
dil3    
- The proportion of germinated grains at each timepoint when A. pollinis diluted 1:3

dil5    
- The proportion of germinated grains at each timepoint when A. pollinis diluted 1:5

dil20    
- The proportion of germinated grains at each timepoint when A. pollinis diluted 1:20

dil100    
- The proportion of germinated grains at each timepoint when A. pollinis diluted 1:100

no_microbes
- The proportion of germinated grains at each timepoint when sterile BK added, no microbes


===================================================================================
Apollinis_benefit_data.csv
- The file is a comma delimited file.

Column headings are in the following order and format:

Treatment_l
- 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
- The tube label, three replicates per treatment

initial count
- The number of cells per mL at the start of the assay for each replicate, as counted on hemocytometer 

final count-24h 
- The number of cells per mL at the 24 hour timepoint for each replicate, as counted on hemocytometer

Date
- The date the assay was started (day of initial count)

===================================================================================

yeast_benefit_data.csv
- The file is a comma delimited file. Data on microbial hemocytometer counts of M. reukaufii, when provided different pollen treatments. 


Column headings are in the following order and format:

Treatment_l
- 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
- The number of cells per mL at the start of the assay for each replicate, as counted on hemocytometer 

24h count
- The number of cells per mL at the 24 hour timepoint for each replicate, as counted on hemocytometer

Date
- The date the assay was started (day of initial count)
===================================================================================

Lowry_data1.csv
- 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    
- Indicates the row of the 96 well plate

column    
- Indicates the column of the 96 well plate 

Timepoint
- 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
    
protein 
- The ug/mL of proetein In the well after correction by dilution factor (4)

Treatment
- Combined pollen and microbe treatment

microbetreatment
- Indicates presence (477) or absence (ctrl) of SCC477

pollentreatment
- Indicates pollen treatment (fresh= germinable pollen,  ungerm= ungerminable, or no pollen)

Date
- Date the assay was started (eg time 0)

===================================================================================

germ_AN.csv
- The file is a comma delimited file. Data on germination and bursting after pollen (germinable or ungerminable) exposure to SCC477 (or no inoculation). 

Treatment
- Combined microbe and pollen treatment: 477= presence of SCC477, ctrl= no inoculation, GP= germinable pollen, UP= ungerminable pollen.

pollentreatment    
- pollen treatment: GP= germinable pollen, UP= ungerminable pollen

Timepoint
- The time at which the measurement was taken relative to when microbes and pollen were combined in the plate.    

image name
- Image name corresponds to 96 well plate well location
    
total
- Total pollen grains in image    

burst
- Number of burst grains in image
    
germ
- Number of germinated pollen grains in image
    
tipburst
- Number of pollen grains that have germinated the burst
    
allgerm    
- combined germinated and tip burst

allburst
- combined tip burst and burst
    
Date
- Date the assay was started (eg time 0)     

**CODE FOR FIGURES AND STATISTICS**

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

**VIDEO**

Video_S1.mp4
- 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. 
- Example of pollen grains bursting after 60m with A. pollinis_SCC477. It is a time-lapse video over 8 minutes. 

**DATA QUESTIONS**
Questions about data and code can be addressed to Shawn Christensen (smchristensen@ucdavis.edu)