To test our hypotheses, samplings for the full root excavation to complete were conducted in an alpine steppe during the growing season (May-September) and nongrowing season (October-April) in the northern Tibetan Plateau, China (30°57′N, 88°42′E), at approximately 4700 m elevation. The site has a continental monsoon climate with a mean annual temperature of 0°C and a mean annual precipitation of 300 mm, occurring mainly during the summer season from June to September. The soil type is cold arid soil, with a mean soil bulk density of 1.39 g cm− 3 and pH c. 8.33. The surface layers (0–1 m) of the soil are seasonally frozen, with freezing initiating from the top in late fall and thawing initiating from the top down in early spring. The native plant community is dominated by Stipa purpruea and Carex moorcroftii and the accompanying species include Artemisia nanschanica and Oxytropis glacialis.
We measured air temperature at 10 cm above the ground using the Decagon ECT sensor fitted with a radiation shield (Decagon Devices, Pullman, Washington, USA). We used Decagon 5TM and EC-TM sensors to measure soil temperature and moisture, integrated across the 10 cm sensor length. In general, the eventual sampling sites can represent the regional native grassland and were > 500 m away from highway to attenuate potential disturbances from traffic or human activities. We elected six alpine steppe species (Stipa purpruea, Carex moorcroftii, Artemisia nanschanica, Leontopodium nanum, Oxytropis glacialis, Astragalus confertus) in the community for measuring their root traits. At each site, at least 15 mature plants that were at a distance of approximately 10 m from each other were selected randomly and excavated in the thaw period (22 March), precomplete thawing period (22 April), mid-complete thawing period (10 August), and freezing period (12 November). Each intact root system was immediately put into polyethylene terephthalate bottles filled with FAA solution for later anatomical measurements. For this study, fresh roots were sliced into 3-cm segments, kept in liquid N2, and transported to the laboratory to determine the activities of various antioxidant enzymes and thiobarbituric acid reactive substances (TBARS).
Based on the root classification (Chen et al., 2001) (Table S2), shaft root was charactered by the well developed primary root and the lateral root successively occurring above it, and a few adventitious roots are also involved in the formation. Creeping rooted plant refers to the vertical downward growth of the primary root, and the horizontal root on the primary root can produce root buds, which form the aboveground branches after reaching the ground, and produce the vertical root downward. Dense cluster type roots are formed by most tiller branches and adventitious roots polymerization. Each tiller node forms only one root and supplies nutrients to its corresponding branch. The root system of rhizome type plants is formed mainly by the abnormal underground organ rhizome. Rhizomes move horizontally in the soil, and then shot from the nodes and give rise to adventitious roots. Meanwhile, in consideration of the root diameter and soil depth distribution, the fine root (Stipa purpruea, Carex moorcroftii, Leontopodium nanum) and shallow rooted (Stipa purpruea, Carex moorcroftii, Leontopodium nanum and Artemisia nanschanica) plants were distinguished.
Prior to the measurements, the root systems were gently washed with deionized water to remove the soils adhering to the roots. After dissection, the whole root was arranged and scanned on a LiDE 220 scanner (Canon LiDE 220) at 600 dpi. Thereafter, the subsamples were oven-dried at 65°C for 48 hours and ground to a fine powder after weighing. The average root diameter, total root length, surface area and volume were determined using the scanned images by WinRHIZO software (Regent Instruments Inc.). SRL was calculated as root total length divided by its dry mass, and RTD was obtained as the ratio of root dry mass to its volume. SRA was defined as the total surface area divided by its dry mass. Briefly, the root samples fixed in FAA were gently washed in deionized water prior to the measurement. The segments were embedded in paraffin individually after dehydration by immersion in a sequence of alcohol solutions. The roots were cut into sections with 8 µm thickness, which were stained with safranine-fast green. The cortex and stele were stained to be green and red, respectively (Kong et al., 2014b). The stained roots were photographed using a camera (NIS Elements D 3.0) in conjunction with the microscope (Nikon, 80i). Stele diameter, cortex thickness and vessel diameter were measured by Image Pro. The number of vessels was counted manually (Long et al., 2013).
Root tissue (1 g) in this study was used for antioxidant enzyme extraction and analyses. The tissues was homogenized in 5 ml 50 mM sodium phosphate buffer (pH 7 for catalase and pH 7.8 for SOD and POD) containing 1% (w/v) polyvinylpyrrolidone and 0.1 mM Na2EDTA. The homogenate was filtered through four layers of cheesecloth and centrifuged at 1500 g for 20 min. After centrifugation, aliquots of the supernatant were passed through a Sephadex G-25 and used to determine the enzyme activities and protein concentration. All extractions were prepared at 0 to 4°C, and enzyme assays were performed at 25°C. The SOD activity was measured spectrophotometrically as described by Beyer and Fricovich (Beyer and Fridovich, 1987). The CAT activity was assayed by the method of Clairborne (Fridovich, 1985). The decomposition of H2O2 was followed by a decline in absorbance at 240 nm for 2 min. One unit of catalase was converted to 1 umol H2O2 min− 1. The POD activity was determined by the method of Chance and Maehly (Machly and Chance, 1955) using guaiacol as an electron donor. The protein concentration in the enzymatic extraction was measured by the method of Stocker (Stocker et al., 2015).
First we divided the herbaceous species into two groups of monocots and dicots based on their root types. Then two-way ANOVA was conducted in SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) to detect the main effects of plant group, growing season/nongrowing season interactions on root morphology and anatomical traits. Least square difference tests were used to conduct post hoc mean comparisons of each parameter in the significant difference test. We used a series of generalized linear mixed effects (GLME) models to test the effects of root anatomical traits. Finally, linear regression was used to analyze the relationship between root morphology and anatomical traits and enzyme activity. Statistical analyses were carried out using the software package R 3.6.3 (R Development Core Team, 2020).