Soil and rice characterisation

Paddy soil and rice grains were obtained from a rice farm near Arbuckle, California (39°01'38.2"N 121°55'36.7"W, elevation: 22 m) in June 2016. Rice grains Oryza sativa L. sub-species japonica, cultivar Calrose, variety M206 were produced in 2015. Paddy soil texture was determined in triplicates at 20 °C by mixing ~75 g of air dry soil with 100 mL of 50 g L−1 sodium hexametaphosphate ((NaPO 3 ) 6 , analytical grade) and topped up to 1 L with water. The fraction of sand was quantified with a hydrometer after 40 s, silt after 2 h, and the remaining fraction was calculated as clay. Soil pH was quantified in triplicates from air-dried soil with double-distilled water at a 1:5 w/v ratio after 2, 24 and 48 h at room temperature. The elemental content of the soil was quantified with X-ray fluorescence (XRF, Spectro XePositive HE XRF Spectrometer, AMETEK, Germany) from 5 g of freeze-dried soil. The cation exchange capacity was quantified in triplicates from air-dried soils with 0.1 M BaCl 2 (analytical grade) at a 1:25 w/v ratio for 4 h at room temperature. Inductively coupled plasma–optical emission spectroscopy (ICP-OES, iCAP6000, Thermo Scientific, UK) was used for quantification of the extracted elements. The total carbon and nitrogen content were quantified in triplicates from freeze-dried soils by combustion in tin foil balls (Carlo-Erba NA 1500, USA). 0.5 M HCl (analytical grade) extractable elemental fraction was quantified with ICP-OES and ferrozine for Fe(II)/Fe(III) speciation64 after extraction of air-dried soil at a 1:40 w/v ratio for 1 h at room temperature. To amend the paddy soil with arsenic, wet field soil was mixed with autoclaved double-distilled water containing 80% arsenite (NaAsO 2 , analytical grade) and 20% arsenate (Na 2 HAsO 4 x7H 2 O, analytical grade) (see Supplementary Table 1). Thus, soil arsenic concentrations varied from a background value of 7.3 mg As kg−1 dry soil to 24.5 mg kg−1. The soil was left to equilibrate for 4 months at the specific climatic condition. Arsenate and arsenite adsorb rapidly to soil mineral surfaces, generally reaching steady-state pore-water concentrations within hours; even with the maturing of arsenic–iron precipitates, steady-state dissolved concentrations are reached within 3 months65,66. Thus, our pre-incubation time allows ample time for arsenic to bind to mineral surfaces and reach conditions similar to field settings.

Greenhouse design

Rice was grown under controlled environmental conditions in growth chambers (polycarbonate, 1.2 × 1.8 × 1.8 m) within two larger greenhouses (Supplementary Fig. 1). Each greenhouse contained four growth chambers: one exhibiting today’s climatic conditions found in Californian rice-growing regions (33 °C (average high daytime temperature during rice-growing season from May to September) and atmospheric CO 2 of 415 ppmv), one with future climatic conditions according to the RCP 8.5 scenario of the latest IPCC report11 (38 °C and atmospheric CO 2 of 850 ppmv) and two growth chambers with either climate parameter (elevated CO 2 or elevated temperature). Outside air was fed into the centre of each chamber through a fan running at ~10 L of air s−1, exchanging the entire atmosphere of the growth chamber ten times per hour. Air exited the growth chamber through a top vent. To achieve doubled atmospheric CO 2 contents in future climate and elevated CO 2 chambers, 99.9% industrial grade CO 2 (Praxair Inc.) was injected continuously directly into the inflowing air at the mouth of the fan. CO 2 flow was monitored in one chamber every second with an infra-red gas analyser (Gashound LI-820, LI-COR Inc.), and adjusted simultaneously for all four high CO 2 chambers using mass flow controllers (Tylan FC-260, International Power Sources Inc.) controlled by a digital to analogue converter (SDM-AO4, Campbell Scientific Inc.). The atmospheric CO 2 content was monitored in all chambers at two locations in between the rice plants using an infra-red gas analyser (LI-6262 CO 2 /H 2 O Analyser, LI-COR Inc.), which was calibrated every second week. CO 2 in chambers with low CO 2 averaged 418 ± 27 ppmv over the season and 885 ± 53 ppmv in chambers with high CO 2 .

The temperature within each chamber was controlled by heater fans (King Electric PHM-1 1500-Watt Portable Milkhouse Heater) connected to timers (digital timer 95205, Chicago Electric); the heaters were operated within the growth chambers from the hours of 05:30 to 19:30.

Each chamber contained two water-filled basins (80 × 60 × 28 cm), in which rice pots were placed. Each water basin was heated constantly by 125 W of fully submersible aquarium heaters (JAGER TruTemp Aquarium Heater, Eheim) to 5 °C lower temperatures compared with the atmospheric daytime temperature (i.e., 27 °C and 33 °C for low and high temperature chambers, respectively). Thus, night-time atmospheric temperatures decreased to 20 °C and 22 °C for low and high temperature chambers between the hours of 19:30 and 05:30. Temperature and atmospheric CO 2 parameters were monitored and controlled with the programme LoggerNet 3.3.1 (Campbell Scientific Inc.), which was executed by the datalogger control unit CR10 (Campbell Scientific Inc.). An even temperature and atmospheric CO 2 distribution within each chamber was assured in pre-experimental climate runs of the greenhouse.

Normal daylight was supplemented with regular lights in the greenhouse between the hours of 06:00 and 19:00 to support naturally occurring sunlight and to minimise shading within each chamber. The photosynthetic photon flux density was between 300 and 600 μmol m−2 s−1 (equivalent to a photosynthetic active radiation of 65–130 W m−2) throughout the day, depending on the prevailing weather during the season.

Experimental design

Each growth chamber hosted microcosms with natural and amended soil arsenic levels, which were placed into either of the two water-filled basins (see Supplementary Fig. 1). The water-filled basins served two purposes; one to provide a lower and more constant temperature to the soil compared with the atmospheric temperature, and second to maintain temperature differences in low and high atmospheric temperatures overnight. Rice pots (high-density polyethylene, 14-cm diameter, 18-cm height) were filled with ~3.4 kg of water-logged paddy soil to 4 cm below the rim. Rhizon samplers (19.21.25, Rhizosphere Research Products) were placed horizontally midway in each pot. Soil was flooded with tap water, pH 7, and irrigated continuously with pumps through a drip irrigation system using water from the basins.

Rice variety M206 was germinated in sterilised tap water under the different climatic conditions. After 1 week of germination, seedlings of similar shoot and root lengths were placed into pots. The soil was fertilised with urea (CON 2 H 4 , analytical grade) using 185 kg nitrogen ha−1 at the beginning of the season, after 4.5 weeks of growth and at panicle initiation, according to practices performed by Californian farmers. Three plants were planted per pot and eight pots per environmental condition, two independently run greenhouses with four climate chambers each. To minimise light, temperature and atmospheric CO 2 biases due to placement of plants within and across chambers, pots were moved around within basins once a week, and chambers were switched every 3–4 weeks.

Pore-water analysis

Pore-water geochemistry was examined regularly by drawing pore-water through rhizon samplers into acid-washed, butyl-stoppered and evacuated glass vials. The pore-water was filtered through 0.2 -µm syringe filters in an anoxic chamber (96% N 2 /4% H 2 atmosphere) and diluted in 2% nitric acid (analytical grade) for total elemental (As, Fe) quantification with ICP-OES. Pore-water organic and inorganic carbon was quantified using the total organic carbon method (TOC) on a Shimadzu TOC-L analyser with in-line acidification (phosphoric acid). Once a month, an aliquot of the unfiltered pore-water was used to determine the pore-water pH, which remained constant during growth (data not shown). Arsenic speciation in the pore-water was analysed using an arsenic speciation cartridge (anion exchanger, Metalsoft, USA) and double-checked by ion chromatography (Dionex ICS-3000, Dionex Corp., USA, PRPX-100 column using a 10–40 mM NH 4 H 2 PO 4 pH 5.6 gradient) coupled to ICP-MS (XSeries2, Thermo Fisher Scientific Inc., USA) (IC–ICP-MS). Using IC–ICP-MS, we found that DMA was present in pore-water, but only contributed to <3% to total arsenic, which was negligible (Supplementary Fig. 7). Thus, we used the simpler arsenic speciation cartridge method, discriminating more easily between charged (arsenate) and non-charged (arsenite) arsenic species. Thereby, DMA and MMA are most likely summarised with arsenate. To do so, 2–3 mL of pore-water were diluted in 13–14 mL of anoxic double-distilled water and run through a water-flushed arsenic speciation cartridge, which retains the charged arsenic (likely mainly arsenate). Uncharged arsenic (arsenite) in the filtered pore-water was quantified with ICP-OES. The amount of arsenate was calculated by subtracting the measured concentration of arsenite from the total pore-water arsenic.

Plant analysis

Plant growth over time was monitored weekly by quantifying plant height, tiller number and panicle number. At harvest, each rice plant was cut-off 2 cm above the water level, packaged into butcher paper and dried at 40 °C in the dark for 4 weeks. Dried plant material was separated into filled spikelets, empty spikelets, panicles and straw (combined leaves and stems), and each fraction was weighed and counted. The yield is defined as the weight of filled spikelets. The harvest index is defined as the grams of filled spikelets produced per plant compared with the grams of vegetative, aboveground tissue produced per plant. Individual grain weight, percentage of filled grains and the number of spikelets per panicle were calculated from weight and count data. Husks were removed from grains by rubbing filled spikelets between two plastic crusher plates. Grains, empty spikelets, husks and green biomass were ground (conventional mechanical grinding followed by liquid nitrogen grinding when needed). Ground grains were stored in the freezer at −80 °C to minimise arsenic speciation changes. Between 0.05 and 0.5 g of different plant tissue was extracted with 3 mL of 65% nitric acid (HNO 3 , analytical grade) and 2 mL of 30% H 2 O 2 (analytical grade) in a microwave digester (CEM Mars 6 Digester) with a 15-min ramp phase to 15 min at 180 °C, followed by a 30 min cool-down phase. Extractants were diluted in double-deionised water, and elemental contents were quantified with inductively coupled plasma mass spectrometry (ICP-MS) (XSeries2, Thermo Fisher Scientific Inc., USA)67,68. For arsenic speciation, 1 g of ground rice grain was extracted with 10 mL of 0.28 M HNO 3 for 90 min at 95 °C in a heating block (Digi PREP Jr, SCP Science, Canada) followed by a 20 min cool-down phase. Extractants were filtered through a 0.45 -µm filter (SCP Science), and arsenic species were separated and analysed by IC–ICP-MS using the method described above for pore-water speciation. The European Reference material ERM-BC211 was used for rice speciation and TMDA-54.4 (Environment Canada) for calibration accuracy. Every 15th sample was spiked with 2 ppb and 5 ppb As(V), As(III), DMA and MMA to check for the recovery of each of these species. The first sample that was analysed was re-analysed at the end of the run to check for species transformation before analysis and signal drift of the instrument. Further, a 10 ppb calibration standard was measured after every 15th sample to check for instrument signal drift. Mean and standard deviations were calculated of biological replicates and unpaired, two-tailed Welch t tests were performed across all soil arsenic and climatic conditions.

Microbial analysis

Rhizosphere soil aliquots were taken sterilely from soil around plant roots midway at the same height as rhizon samplers were placed within the pots, shock-frozen in liquid nitrogen, and stored at −80 °C. The total DNA was extracted from 0.25 g of two frozen samples and using the PowerSoil® DNA Isolation Kit (Qiagen, Germantown, MD, USA). The quality and quantity of DNA were verified on 1% (w/v) ethidium-bromide-died agarose gels and fluorometric quantification with Qubit® 2.0. The 16S rRNA gene copy numbers of the total Bacteria were amplified and quantified with qPCR using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a StepOneTM Plus cycler (Applied Biosystems, Waltham, MA, USA). As a standard, a 16S rRNA gene fragment from Gemmatimonas sp. was amplified from the rhizosphere DNA extracts with general bacterial primers GM3 (8F, (5′-AGAGTTTGATCMTGGC-3′)69 and 1392-R (5′-GACGGGCGGTGTGTRCA-3′)70 and cloned into the plasmid vector pGEM®-T easy (Promega Corporation, Madison, WI, USA). In a 10 µL of reaction volume, 1 µL of 100-fold diluted DNA extract or a tenfold dilution series of the standard plasmid DNA were used with 1× SsoAdvanced Universal SYBR Green Supermix, 75 nM of primer 341-F (5′-CCTACGGGAGGCAGCAG-3′)71 and 225 nM of primer 797-R (5′- GGACTACCAGGGTATCTAATCCTGTT-3′)72. The qPCR programme ran with 3 min at 98 °C, 40 cycles of 15 s at 98 °C and 30 s at 60 °C, and followed by melting curve analysis. The data analysis was performed using the StepOneTM 2.3 software. Each of three independent DNA extractions were measured in triplicates.

X-ray fluorescence imaging

The distribution of arsenic and other elements across husked rice grains was visualised with X-ray fluorescence (XRF) mapping using beamline 2–3 at Stanford Synchrotron Radiation Lightsource (SSRL). The beamline receives X-rays from a 1.3 Tesla Bend Magnet, and it is equipped with a double-crystal Si (111) monochromator for energy selection, a vortex silicon drift detector and ionisation chambers. The sample is positioned in a 45° angle to the incoming beam and another 45° angle to the detector (90° angle between incoming beam and detector). The Kirkpatrick-Baez mirror system achieves a beam size of ~2 × 2 microns. The beam was calibrated to the arsenic K-edge of an arsenate standard, and maps were run at the energy for arsenite), dimethylarsinic acid (DMA) and total arsenic, rastering at a 0.013-mm step size and a 45-ms dwell time. Mapping energies for arsenite and DMA were chosen as they contribute >95% to the respective inorganic and organic arsenic fractions in the grain (previously verified with IC–ICP–MS in grain extracts). Rice grains were split in half on the longitudinal axis of the grain and fixed on glass slides. One single replicate grain was run per climatic and soil arsenic condition. Using the software SMAK73, arsenic fluorescent counts were quantified to an arsenic standard of known concentration (47 µg As cm−2) and expressed in mg As kg−1 grain for a sample thickness of 1 mm and a grain density of 0.8 kg of grain L−1. Average fluorescent counts for DMA and arsenite were obtained for husks, brans and endosperm. DMA and arsenite ratios from the husk to bran, bran to endosperm and within husk, bran and endosperm were calculated.

Data analysis

Mean and standard errors were calculated of all data set. The data were verified for normal distribution with the Kolmogorov–Smirnov Test. Standard Student’s t tests were combined with a two-factorial ANOVA analysis to display significant differences in means of data and illuminate significant interactions between climatic condition and soil arsenic (Supplementary Table S2).