Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Remediation and Control Technologies
The Role of Vertical Transmission of Shoot Endophytes in Root-Associated Microbiome Assembly and Heavy Metal Hyperaccumulation in Sedum alfredii Jipeng Luo, Qi Tao, Radek Jupa, Yuankun Liu, Keren Wu, Yuchao Song, Jinxing Li, Yue Huang, Linyun Zou, Yongchao Liang, and Tingqiang Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01093 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on June 4, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Environmental Science & Technology
1
TITLE
2
The Role of Vertical Transmission of Shoot Endophytes in Root-Associated
3
Microbiome Assembly and Heavy Metal Hyperaccumulation in Sedum alfredii
4 5
Corresponding author: Tingqiang Li, Ph.D. and Professor, College of Environmental and
6
Resource Sciences, Zhejiang University, Hangzhou 310058, China; Tel: +86-571-88982518,
7
Fax: +86-571-88982907, E-mail:
[email protected] 8
Jipeng Luo,*† Qi Tao,*‡ Radek Jupa,§Yuankun Liu,† Keren Wu,† Yuchao Song,†
9
Jinxing Li,† Yue Huang,† Linyun Zou,† Yongchao Liang,† Tingqiang Li†
10
† Ministry of Education Key Laboratory of Environmental Remediation and Ecological
11
Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou
12
310058, China.
13
‡College of Resources, Sichuan Agricultural University, Chengdu 611130, China
14
§Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská
15
2, 61137 Brno, Czech Republic.
16
*These authors contributed equally to this work
17
Author contributions: J.L., Q.T. and T.L. designed the experiments; J.L., Q.T., Y.L., Y.H.,
18
L.Z., K.W., J.L. and Y.S. performed the experiments; J.L. and Q.T. analyzed the data; J.L.,
19
Q.T., R.J.,Y.L., and T.L. wrote the manuscript with contribution of all of the coauthors; T.L.
20
supervised the experiments and writing of the article.
21 22
Conflict of interest: The authors declare that they have no competing interests.
23
1
ACS Paragon Plus Environment
Environmental Science & Technology
25
ABSTRACT: The transmission mode of shoot-associated endophytes in hyperaccumulators
26
and their roles in root microbiome assembly and heavy metal accumulation remains unclear.
27
Using 16S rRNA gene profiling, we investigated the vertical transmission of
28
shoot-associated endophytes in relation to growth and Cd/Zn accumulation of Sedum alfredii
29
(Crassulaceae). Endophytes were transmitted from shoot cuttings to the rhizocompartment
30
of new plants in both sterilized (γ-irradiated) and native soils. Vertical transmission was far
31
more efficient in the sterile soil and the transmitted endophytes have become a dominant
32
component of the newly established root-associated microbiome. Based on 16S rRNA genes,
33
the vertically-transmitted taxa were identified as the families of Streptomycetaceae,
34
Nocardioidaceae, Pseudonocardiaceae, and Rhizobiaceae. Abundances of Streptomycetaceae,
35
Nocardioidaceae and Pseudonocardiaceae were strongly correlated with increased shoot
36
biomass and total Cd/Zn accumulation. Inoculation of S. alfredii with the synthetic bacterial
37
community sharing the same phylogenetic relatedness with the vertically transmitted
38
endophytes resulted in significant improvements in plant biomass, root morphology, and
39
Cd/Zn accumulation. Our results demonstrate that successful vertical transmission of
40
endophytes from shoots of S. alfredii to its rhizocompartment is possible, particularly in soils
41
with attenuated microbiomes. Furthermore, the endophyte-derived microbiome plays an
42
important role in metal hyperaccumulation.
2
ACS Paragon Plus Environment
Page 2 of 34
Page 3 of 34
44
Environmental Science & Technology
Table of Contents Graphic
45
3
ACS Paragon Plus Environment
Environmental Science & Technology
47
1. INTRODUCTION
48
Land plants harbor a diverse array of bacterial microbiota on and inside their tissues.
49
These microbiota affect plant growth and health, and therefore success in a large variety of
50
ways.1,2 It is well known that plants rely on their endophytic microbiota for nutrient
51
acquisition,3 growth promotion,4 suppression of phytopathogens and stress tolerance.5,6
52
However, the wider role of the microorganisms, particularly the role of the shoot-associated
53
(leaf and stem) microbiota rather than the well-studied root-associated bacterial assemblages,
54
on plant success is still being unraveled.7-9 Furthermore, endophytic bacteria are likely to be
55
highly important symbionts affecting the host plants interaction with the abiotic and biotic
56
environments. These endosymbionts establish an intimate symbiosis within plant tissues and
57
their survival and reproduction depends largely on the plant.10 In this case, the success of the
58
endophyte is coupled with the success of the host.
59
The transmission mode is an important feature of endophytes. Generally, the endophytic
60
microbiome may be assembled either through horizontal transmission from the environment
61
to host and/or via vertical transmission from parent to progeny.9,11 It is commonly
62
considered that root endophytes are predominantly assembled via horizontal transmission
63
(recruitment of microorganisms from the surrounding soil).7-9 However, numerous studies
64
have recently confirmed the existence of vertical (seed-based) transmission of endophytes in
65
plant species such as rice,12 Brassicaceae13 and some representatives of grasses.14 These
66
studies highlighted that seed endophytes could migrate to the newly established root
67
endosphere or rhizosphere compartment. However the mechanisms and factors affecting the
68
vertical transmission of endophytes remain elusive for most host-endophyte associations. In
69
addition, vertical transmission of endosymbionts has not been described in hyperaccumulator
70
species.
71
Endophytic bacteria can alter the interactions of plants with metals in the environment,
4
ACS Paragon Plus Environment
Page 4 of 34
Page 5 of 34
Environmental Science & Technology
72
and this trait can be used to maximize the potential for phytoremediation of metal
73
contaminated sites.15 However, the maintenance of the appropriate endophytic taxa by the
74
host plant and their inheritance to following plant generations are important prerequisites for
75
the long-term success
76
Cd-hyperaccumulator Sedum alfredii (Crassulaceae) found that root microbiomes were
77
primarily derived from the taxa resident in the soil surrounding roots rather than vertically
78
transmitted as seed-borne endophytes.16 This implies that endophytes present in the seed of S.
79
alfredii may not be successful competitors against soil microorganisms, resulting in failure
80
in their transmission to the new generation of plants. Nevertheless, shoot cuttings, separated
81
from hyperaccumulators grown on metal contaminated sites, were shown to carry large
82
amounts of endophytes with great potential to survive in metal-stress conditions.17 The
83
recruitment of these endophytes altered plant growth characteristics and heavy metal
84
hyperaccumulation.18 Therefore, vertical transfer of endophytes (and the traits they confer)
85
via shoot cuttings is likely possible for some hyperaccumulator species. However, direct
86
evidence supporting this hypothesis is unavailable yet. Moreover, a handful of experimental
87
studies have illustrated that the vertically-transmitted seed endophytes of rice and bean could
88
propagate as part of the new plant microbiota,12,19 indicating that the seed-borne
89
endosymbionts were involved in root-associated microbiome assembly. However, whether
90
shoot endophytes of hyperaccumulators will become important founders of the newly
91
established root microbiomes via vertical transmission, remains unknown.
92
of
this
function.
For
example,
experiments
using
the
Under metal stress, endophytic bacteria can promote plant growth by producing
93
phytohormones,
increasing
nutrient
solubility
94
1-aminocyclopropane-1-carboxylate (ACC) deaminase,1,15 as well as alleviate the damages
95
of heavy metals to plants by altering the bioavailability/or toxicity of heavy metal.15,18 Given
96
the extent of plant-endophyte associations and physiological processes involved, it is not
5
ACS Paragon Plus Environment
or
secreting
Environmental Science & Technology
97
surprising that considerable number of researches have been conducted with goal of utilizing
98
endophytic bacteria to optimize phytoremediation systems and enhance their use.20,21
99
However, most empirical studies on the functions of endophytes have implemented
100
inoculation–based transfer of the microbiome within artificial cultivation (laboratory)
101
systems; these do not generally consider mechanisms of vertical transmission.
102
S. alfredii is well known as an important Zn/Cd co-hyperaccumulator that can be easily
103
propagated using shoot cuttings.22,23 In this species, cultivation of shoot cuttings has been
104
used to investigate mechanisms of Cd/Zn uptake, xylem transport, and Cd/Zn tolerance.24,25
105
Studies indicated that S. alfredii are host to a diversity of metal-tolerant endophytes that
106
could promote plant growth and heavy metal accumulation.16,21 However, the
107
community-level dynamics of shoot endophytic microbiome composition and their effects on
108
root microbiome acquisition and assembly in natural conditions remains unknown. In this
109
study, using S. alfredii as a model plant, we investigated the transmission of endophytic
110
bacteria from shoot cuttings to root-associated compartments, and examined the effects of
111
the newly established microbiomes on plant growth and metal accumulation.
112
2. MATERIALS AND METHODS
113
2.1. Plant and Soil Collection. Mature plants of S. alfredii were collected in an old
114
Pb/Zn mining area in Quzhou city, Zhejiang province, China. Simultaneously with plants,
115
top soil (10 cm) were collected, and then were air dried, sieved (< 2 mm) and separated into
116
two parts. One part was exposed to gamma ray irradiation (30 kGy; γ-irradiated soil), while
117
the other part was untreated (native soil). Basic physicochemical properties of this soil
118
(Table S1) were determined according to the previously described standard procedures.16
119
For plant cultivation, shoot cuttings with similar size and comparable leaf numbers were
120
separated from mother plants. The newly exposed (cut) surfaces of the offshoot clones were
121
sterilized in 70% (v/v) ethanol for 30s, 3% (w/v) sodium hypochlorite solution for 150s, and
6
ACS Paragon Plus Environment
Page 6 of 34
Page 7 of 34
Environmental Science & Technology
122
then washed six times with sterile water.
123
2.2. Experimental Design, Plant and Microbiota Cultivation. The γ-irradiated and
124
native soils were used to investigate the vertical transmission of shoot endophytes by shoot
125
cuttings propagation. Subsequently, the hydroponic and tissue culture experiments were
126
conducted, by inoculating S. alfredii with the candidate vertically-transmitted bacterial
127
isolates (Table S2), to assess the effects of these bacteria on plant growth, root development
128
and heavy metals accumulation. For each of these three cultivation experiments, plants were
129
grown under standard growth-chamber conditions: 16-h photoperiod, 300 μmol m−2s−1 of
130
average light intensity, 26/20°C of day/night temperature, 75% of relative humidity.
131
For soil cultivation, shoot cuttings of S. alfredii were surface-sterilized and then
132
transplanted into the ‘rhizosphere compartment’ of a rhizobox filled with either 2.5 kg of
133
γ-irradiated or native soil.26 The ‘non-rhizosphere chamber’ of rhizobox (without plants) was
134
treated as bulk soil. Pots without plants were defined as unplanted treatment. Each treatment
135
had five replicates. The soil was moistened with sterile water and gravimetrically adjusted
136
every 4 days to 60% of water holding capacity. After four months of cultivation, shoot
137
biomass and accumulation of heavy metal were determined as described in the
138
Supplementary Information. The collection of bulk soil, rhizosphere soil, and root
139
endosphere samples was performed as described in a previous study.16 The collected samples
140
were immediately frozen in liquid nitrogen and stored at -80℃ until subsequent analysis.
141
For hydroponic cultivation, S. alfredii shoot cuttings were surface-sterilized and
142
cultured in axenic nutrient solution24 either with or without 25μM Cd(NO3)2 and 500μM
143
ZnSO4. The nutrient solutions were inoculated with a synthetic microbial community
144
consisting of four endophytic bacterial isolates (strains SaSS1, SaSS28, SaSS40, and SaSS46;
145
Table S2) originating from the shoot of S. alfredii plants. These strains share a similar
146
phylogenetic relatedness with the potential vertically-transmitted endophytes and show high 7
ACS Paragon Plus Environment
Environmental Science & Technology
147
resistance to Cd and Zn (details of the acquisition of bacterial inocula are provided in
148
Supplementary Information). Treatments included Control (pure axenic nutrient solution),
149
Cd2+ and Zn2+ treatment (HM), and a combination of HM and bacterial strains (HM+Bact).
150
Each treatment was replicated four times, and plants were harvested after 60 days.
151
Tissue-based cultivation was performed to investigate the effects of the four
152
aforementioned bacterial strains on root morphology and plant development. Briefly, stem
153
offshoots were cut transversely into 0.5 cm segments and transferred to MS medium
154
solidified with 0.7% agar. The experiment comprised four treatments: (i) Pure MS medium
155
(Control); (ii) MS medium containing 5μM Cd(NO3)2 and 50μM ZnSO4 (HM); (iii) MS
156
medium contains four bacterial strains (1×107of total bacterial cell numbers g-1 agar) (Bact);
157
and (iv) MS medium containing 5μM Cd(NO3)2 and 50μM ZnSO4 and four bacterial strains
158
(HM+Bact). Root length and plant biomass were measured after two months of cultivation.
159
2.3. DNA Extraction, Quantitative Real-Time PCR (qPCR), and Amplification
160
Sequencing. The stem and leaf samples at the time of transplantation, and root samples
161
after four months of soil cultivation were mechanically homogenized. DNA was extracted
162
from the homogenized tissues, along with unplanted, bulk, and rhizosphere soils. Similarly,
163
RNA was extracted from the root samples from the hydroponic experiment using a
164
commercially available kit. The total bacterial cell numbers and relative expression of root
165
transporter genes (Table S3) were determined by qPCR. Extraction procedures and the
166
protocol of qPCR are described in detail in the Supplementary information.
167
In order to capture the true diversity of bacterial communities in the shoot- and
168
root-associated compartment samples, the V5-V7 and V3-V4 regions of the 16S rRNA gene
169
were amplified, respectively. 27 The DNA extracted from soil and root samples was used as
170
template for amplification of the 16S rRNA gene V3-V4 region with the primer set
171
S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21.28 For the leaf and stem DNA samples,
8
ACS Paragon Plus Environment
Page 8 of 34
Page 9 of 34
Environmental Science & Technology
172
the 16S rRNA gene V5-V7 region was amplified with the primer set 799f and 1193r,29,30
173
selected as it shows very low amplification of non-target DNA (e.g. chloroplast and
174
mitochondria) and retrieves a high number of bacterial reads.27. Each sample was amplified
175
with a primer set link a dual-index barcode. This enabled sequencing of pooled (equimolar)
176
amplicons (MiSeq; Illumina) and downstream assignment of amplicons to treatments based
177
on the unique barcodes. Sequence data were submitted to the NCBI Sequence Read Archive
178
under Bioproject PRJNA432836 and BioSample accession numbers from SAMN08462835
179
to SAMN08462884.
180
2.4. Sequence-Data Processing and Analyses. A total of 1,639,312 sequences was
181
obtained after quality control, with a median read count per sample of 29,284 (range:
182
22,368-83,117). The bioinformatic analyses of raw sequence data including de-multiplex,
183
quality control, and taxonomy classification were performed using the QIIME2 (v.2018.8)
184
software.31 The DADA232 method was used to perform sequence quality control, in which
185
the reads were denoised into amplicon sequence variants (ASVs), and then a feature table
186
was generated. Taxonomic classification of the denoised sequences was performed by
187
training the naive Bayse classifier using Silva 132 99% OTUs (Operational Taxonomic
188
Units) full-length reference sequences. Additionally, the ASV table was normalized by
189
rarefying to 8000 reads per sample to reduce the bias of different sequencing depth between
190
samples. For detailed description of sequence processing, see the Supplementary
191
Information.
192
2.5. Fluorescence in Situ Hybridization (FISH). We applied FISH to visualize bacteria
193
and Actinobacteria within S. alfredii stem, root and rhizopshere soil samples according to a
194
previously described protocol.33
195
2.6. Statistical Analyses. The statistical analyses were mainly performed in R software
196
(http://www.r-project.org) using the sequence data set of 5331 ASVs across 50 samples, and
9
ACS Paragon Plus Environment
Environmental Science & Technology
197
the detailed descriptions of individual analyses are provided in the Supplementary
198
Information.
199
3. RESULTS
200
3.1. Plant Growth and Heavy Metal Accumulation. S. alfredii growth was
201
significantly promoted when cultivated in γ-irradiated soil (Figure 1a, b). The fresh weight
202
(FW) and dry weight (DW) of plant shoots cultivated in γ-irradiated soil were significantly
203
increased by 104% (P=0.005) and 122% (P=0.015) compared with plants grown in the
204
native soil (Figure1c). The concentrations of Cd and Zn in shoots were also higher in plants
205
grown in the γ-irradiated soil than in the native soil (Figure 1d). Specifically, S. alfredii
206
extracted 1389.1 mg Cd and 25064.2 mg Zn kg-1 dry shoot biomass from the γ-irradiated soil
207
at the harvest, which were 4.6-fold and 4.3-fold higher than that from the native soil,
208
respectively.
209
In hydroponic experiment, bacterial inoculation significantly (P
