Legacy effects of historical grazing alter leaf stomatal characteristics in progeny plants

Field site and plant material

The experimental materials were taken from an artificial grassland located at the field station of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences, located in Hohhot, Inner Mongolia, China (N: 40°36′; E: 111°45′; H: 1,065 m). The artificial grassland was established for the field research in July 2009, and planted with the forage species L. chinensis, Elymus nutans Griseb., and Medicago sativa L. cv. Aohan., Emanmus sibiricus L., etc. The two treatments included overgrazing (GZ) and no grazing (NG) in this grazing experiment plot, three replications were conducted for each treatment, and the area of each plot was about 0.64 hm². For grazing treatments, six sheep continuously grazed in the GZ plot during the growing season from May to October each year at a stocking rate of about nine sheep per hectare. This study selected the rhizome buds of L. chinensis as offspring generation to examine the legacy effects of historical grazing in parental plants.

Greenhouse experiment

Fresh grass had not germinated at the early spring of 2018, and the rhizome buds of L. chinensis were randomly taken from both GZ and NG plots in the artificial grassland. The rhizomes coming from different parental plants were cut to 2–3 cm lengths and each rhizome with a bud was planted into a pot and transferred to the greenhouse for cultivation. The buds were kept in the greenhouse for 7 weeks. Soil was taken from the grazing experiment plots, and was sieved and mixed to ensure uniformity of the soil. The rhizomes were planted into pots with a diameter of 18 cm after the pots were filled with soil. Each treatment used seven pots with three rhizomes per pot. In total, 14 pots were randomly arranged in the greenhouse. The temperature was about 25 °C during the day and 15 °C at night. The phenotypic traits, gas exchange parameters, stomatal traits, and related gene expression studies were successively tested after plant transplantation based on the following methods.

Morphological traits

The plant height was measured from the 2nd week after transplantation and was continually tested every week. The leaf length (LL) and leaf width (LW) of the second leaf from the top of each individual were measured at week seven of growth in the greenhouse. The leaf area was calculated according to the formula: Area = 0.655* (LL × LW) (Yajun, 2009).

Gas exchange measurements

After about 7 weeks of culture, the gas exchange parameters of the second leaf from the top of each plant were measured using a LI-6400 (LI-COR, Lincoln, NE, USA) photosynthetic measurement system during a clear morning from 9:00 to 11:00. Measurement indicators included: net photosynthetic rate (P_N), transpiration rate (E), inter-cellular CO₂ concentration (Ci), stomatal conductance (gs), and water use efficiency (WUE) calculated from photosynthetic rate and transpiration rate (WUE = P_N/E). The gas exchanges were measured as photosynthetically active radiation (PAR) of 1000 ± 10 μmol m⁻² s⁻¹ and CO₂ concentration of 350 ± 3 ppm.

Stomatal traits

After about 7 weeks of culturing, the second leaf of each of the treated plants of L. chinensis was collected to determine the stomatal traits using the impression approach (Xu & Zhou, 2008). After collection, the leaves were rapidly fixed with 2.5% glutaraldehyde, and brought back to the laboratory. The abaxial and adaxial epidermis of the leaf were first cleaned using a paper towel, and then carefully smeared with transparent nail polish in the tail, middle, and top area, gently peeled off after approximately 15 min, then immediately mounted on a glass slide, covered with a cover slip, and finally observed and photographed using an optical microscope (BX53, Olympus, Tokyo, Japan). To count the number of stomata in adaxial and abaxial leaf surfaces in the microscope at 40× magnification, and to calculate the stomatal density (stomatal density = number of stomata/area in view), five fields of view were randomly observed for each piece. Seven stomata were randomly selected from the microscopic slides, and stomatal length and width in the microphotographs were analyzed with Image J 1.0 image processing software (National Institutes of Health, Bethesda, MD, USA). Since the degree of opening of the pores changes constantly, the length and width of guard cells were selected to represent the maximum degree of opening of the pores, that is, the size of the pores (Fig. S1).

Real-time PCR analysis

Samples were taken from the second or third leaf of offspring plants treated with enclosure and overgrazing of parental plants after about 7 weeks of culture, then stored at −80 °C. RNA isolation used the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) following the procedures provided by the manufacturer. Then, the quantity of RNA was evaluated by electrophoresis on a 1.0% agarose gel, and the concentration was measured by an ultraviolet spectrophotometer (UV-2550PC, Shimadzu, Japan). High-quality total RNA samples were used for subsequent experiments. According to the manufacturer’s instructions, any possible DNA contamination was eliminated from RNA samples, and first-stand cDNA was synthesized by using the Prime Script ^RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). The cDNA samples were stored at −20 °C for real-time PCR analysis. In addition, eight differentially expressed genes related to stomatal development and regulation were selected based on transcriptome data (Ren et al., 2018). The program primer premier 5 was used to design the required primer sequences, and the primer details of these genes are presented in Table S1 of the Supporting Information. The real-time PCR for gene expression was performed on Quant Studio™ 6 Flex System Software using SYBR Green 1 reagents, following the instructions of test kits.

Statistical analyses

All statistical analyses were conducted in Microsoft Excel, and independent samples T tests were performed to assess the differences between treatments for phenotypic characteristics, photosynthetic parameters, and stomatal traits at the P < 0.01 and P < 0.05 levels of significance using SPSS 19.0 statistical software (Systat Software Inc., Chicago, IL, USA). Graphical representations were generated with Sigmaplot 13.0 software (Systat Software Inc., San Jose, CA, USA).

The effects of historical grazing on stomatal characteristics

Plants can alter stomatal activity to cope with environmental stresses such as drought (Changhai et al., 2010), cold (Reynolds-Henne et al., 2010), insects (Papazian et al., 2016), and even grazing (Zhang, 2010). This study provides clear evidence that the grazing history can impress legacy effects on traits and functions of leaf stomata in L.chinensis. After grazing, stomatal density increased significantly on the basal of adaxial and abaxial leaf surfaces and decreased significantly on the middle of abaxial leaf surfaces. Similar findings have been reported for Thalictrum aplinum, Kobresia humilis, Gentiana straminea, and Elymus nutans Griseb (Zhang, 2010). Moreover, stomatal length of the progeny developed from grazed parents increased, while stomatal width decreased significantly in abaxial leaf surfaces. These data suggested that stomata of progeny plants tended to close because of the long-term overgrazing experience of their parental plants, which resulted in a decrease in gs. A similar result of stomatal closure caused by herbivory was also reported previously (Papazian et al., 2016). Hence, it may be an alternative strategy to close the stomata to adapt to grazing stress.

Stomata are deliberately regulated by a series of genes to help plants adapt to environmental stimuli. This study investigated eight key genes that play key roles in the development and movement of stomata. It has been suggested that historical grazing affects the expression level of these genes on the progeny developed from grazed parents. After long-term grazing, the expression of seven of these eight tested genes, mainly related to stomatal development and regulation, were significantly decreased. For example, ABCCL4 and ABCG5 are two members in the superfamily of the ATP-binding cassette (ABC) transporters, which has more than 120 members in both rice (Oryza sativa) and A. thaliana. Stomatal regulation is one of the fundamental processes in which ABC transporters participate (Rea, 2007). A previous study supported that ABC transporter AtMRP5 mutants tended to have more closed stomata than wild-type control plants, resulting in decreased transpiration rate and increased drought tolerance (Klein et al., 2003). Therefore, the down-regulation of the expressions of ABCCL4 and ABCG5 may also be relevant to closing stomata. In addition, GNAT1 plays an important role in signal transmission, as G proteins participate in ethylene-induced stomatal closure and functions through hydrogen peroxide synthesis in A. thaliana (Ge et al., 2015). Moreover, in this study, three cell-surface-resident receptor-like kinases genes (ER1, ER, and ERL1) were detected, which encode members of the large family of plant LRR receptor-like kinases (RLKs) (Shpak et al., 2004), which play a critical role in the regulation of stomatal density. Shpak (2005) identified the ER-family LRR-RLKs as negative regulators of stomatal development, and the down-regulation of ER genes and the up-regulation of CYCP3L-1 provide molecular evidence for increased stomatal density and length on the basal of adaxial and abaxial leaf surfaces. Another serine/threonine–protein kinase gene (SAPK10), which is specifically expressed in guard cells, plays an important role in stomatal movement (Min et al., 2019). However, the regulation mechanism of SAPK10 remains unclear and requires further study.

Legacy effects on stomata

Stomatal numbers and their pattern on the leaf surface can be either directly or indirectly influenced by the growth environment. Stomatal density is one of the key factors that may regulate the stomatal conductance and functions (Doheny-Adams et al., 2012). Increased stomatal density offers more potential for CO₂ diffusion into the leaf, which facilitates higher rates of gas exchange in responses to environmental stimuli (Yoo et al., 2011; De Boer et al., 2016). Furthermore, a close positive correlation exists between stomatal conductance and photosynthetic rate (Buckley & Mott, 2013; Farquhar & Sharkey, 1982). Tanaka et al. (2013) investigated A. thaliana mutants with altered stomatal density and showed that the photosynthetic rate could be enhanced by 30% via increasing stomatal density in STOMAGEN-overexpressing A. thaliana compared with wild-type plants. In the present research, the photosynthetic rate decreased significantly in response to long-term grazing treatment in the parental generation of L. chinensis. A similar finding was also reported in previous research (Ren et al., 2017). The performance is the same in the subsequent generation that developed from grazed parents. However, the photosynthetic rate of offspring plants decreased by 28.6% compared with that of the un-grazed control. The possible reason is that stomatal aperture weights much than stomatal density on which gs is determined (Weyers & Lawson, 1997). Furthermore, changes in stomatal density can be compensated for by changes in stomatal opening or closure. For example, Büssis et al. (2006) used transgenic A. thaliana plants that over-expressed the SDD1 gene and sdd1-1 mutants showed that increased stomatal density could be compensated for by decreased stomatal aperture.

Many studies have showed that livestock grazing may change soil quality or properties and leads to soil compaction relative to ungrazed soil (Daniel et al., 2002; Drewry, Cameron & Buchan, 2008; Kotzé et al., 2013; Pulido et al., 2016; Byrnes et al., 2018). This in turn can result in decreased soil pore space, reduced infiltration, and even decrease the supply of available water (Willatt & Pullar, 1984; Kotzé et al., 2013; Tate et al., 2004). To address the water limitation, plants may decrease their stomatal aperture to minimize water loss. However, the closure of stomata may also decrease the uptake of CO₂, which may decrease the photosynthetic rate and inhibit plant growth. Hence, plants may face the trade-off between water loss and CO₂ uptake. For example, Case & Barrett (2001) investigated two populations of Wurmbea dioica separately in wet and dry conditions, their results showed that the stomata of the dry populations were partly closed to minimize water loss and decrease photosynthesis rates. The detailed mechanisms of the stomatal response to grazing are not easy to explain, because grazing involves a complex set of factors. However, the results of the present study suggested the existence of legacy effects of livestock grazing in the parents with regard to the stomatal behavior in offspring generation of L. chinensis. Moreover, the changes in stomatal characteristics decreased stomatal conductance and photosynthesis rate, which resulted in a dwarf phenotype of offspring plants (Fig. 6). So far, exceedingly little is known about the detailed mechanisms of these legacy effects; however, increasing evidence suggests that epigenetic mechanisms such as DNA methylation, histone modifications, and small RNAs contribute to these legacy effects (Agarwal, 2001; Champagne, 2008; Richards, 2011; Boyko et al., 2010). Further research is needed to fully understand the epigenetic mechanisms of how the legacy effects are induced by grazing from the parents to progeny plants.

Legacy effects of historical grazing on progeny plants

Long-term overgrazing, which can affect the stability of the plant community by altering the growth of individual plants (Niu et al., 2015; Zuo et al., 2018), is an important cause for grassland degradation (Bai et al., 2012). Previous research has shown that transgenerational morphological plasticity induced by overgrazing heavily involves photosynthetic function in L. chinensis (Ren et al., 2017). In general, phenotypic plasticity refers to the ability of organisms to modify their morphology, physiology, or behavior and adapt to changing environmental conditions (Wadgymar & Austen, 2019). This experiment further observed that overgrazing experienced by the parental generation could alter stomatal characteristics of the offspring of L. chinensis. The decreased photosynthesis and the dwarfed phenotype may be attributed to transgenerational stomatal plasticity. However, little is known about transgenerational effects induced by overgrazing in forage grass. According to a recent study by Kafle, Wurst & Dam (2019), single herbivory in the parental generation did not prime the transgenerational response, only sequential above- and below- ground herbivory did. Therefore, it can be speculated that transgenerational responses require a certain threshold of environmental stress. Moreover, the developmental stage at which the parental generation is subjected to stress can also influence the performance of their offspring (Burton & Metcalfe, 2014). However, for perennial asexual plants, the threshold of cross-generational effects and how long it can last in the offspring generation remain unknown and require further exploration.

Transgenerational legacy effects have received increasing attention in recent years. Although only the legacy effects raised from historical grazing in L. chinensis was investigated in this study, the results still provide hints what may happen in other plants. Holeski, Jander & Agrawal (2012) have also summarized several examples of transgenerational effects of the resistance to herbivores and pathogens in 11 plant species. Previous studies have shown that the offspring generation could perform better in the maternal than in the non-maternal environment (Galloway & Etterson, 2007; Latzel et al., 2014). L. chinensis, as an asexual plant, consists of many interconnected ramets, where each ramet may experience different environmental conditions. This leads to better adaptation of offspring ramets to the living environment by inheriting information from the maternal generation. Therefore, the legacy effects of historical grazing are more conducive to both the survival and growth of progeny plants.