Physiological, biochemical and phytohormone responses of Elymus nutans to α-pinene-induced allelopathy.

The α-pinene is the main allelochemical of many weeds that inhibit the growth of Elymus nutans, an important forage and ecological restoration herbage. However, the response changes of α-pinene-induced allelopathy to E. nutans is still unclear. Here, we investigated the physiological, biochemical and phytohormone changes of E. nutans exposed to different α-pinene concentrations. The α-pinene-stress had no significant effect on height and fresh weight (FW) of seedlings. The water-soluble proteins, the soluble sugars and proline (Pro) strengthened seedlings immunity at 5 and 10 µL L-1 α-pinene. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased at 5 µL L-1 α-pinene to resist stress. APX reduced the membrane lipid peroxidation quickly at 10 µL L-1 α-pinene. The high-activity of peroxidase (POD), APX along with the high level of GSH contributed to the cellular redox equilibrium at 15 µL L-1 α-pinene. The POD, glutathione reductase (GR) activity and glutathione (GSH) level remained stable at 20 µL L-1 α-pinene. The changes in antioxidant enzymes and antioxidants indicated that E. nutans was effective in counteracting the harmful effects generated by hydrogen peroxide (H2O2). The α-pinene caused severe phytotoxic effects in E. nutans seedlings at 15 and 20 µL L-1. Endogenous signal nitric oxide (NO) and cell membrane damage product Pro accumulated in leaves of E. nutans seedlings at 15 and 20 µL L-1 α-pinene, while lipid peroxidation product malondialdehyde (MDA) accumulated. The chlorophylls (Chls), chlorophyll a (Chl a), chlorophyll b (Chl b) content decreased, and biomass of seedlings was severely inhibited at 20 µL L-1 α-pinene. The α-pinene caused phytotoxic effects on E. nutans seedlings mainly through breaking the balance of the membrane system rather than with reactive oxygen species (ROS) productionat 15 and 20 µL L-1 α-pinene. Additionally, phytohormone levels were altered by α-pinene-stress. Abscisic acid (ABA) and indole acetic acid (IAA) of E. nutans seedlings were sensitive to α-pinene. As for the degree of α-pinene stress, salicylic acid (SA) and jasmonic acid (JA) played an important role in resisting allelopathic effects at 15 µL L-1 α-pinene. The ABA, Zeatin, SA, gibberellin 7 (GA7), JA and IAA levels increased at 20 µL L-1 α-pinene. The α-pinene had a greatest impact on ABA and IAA levels. Collectively, our results suggest that E. nutans seedlings were effective in counteracting the harmful effects at 5 and 10 µL L-1 α-pinene, and they were severely stressed at 15 and 20 µL L-1 α-pinene. Our findings provided references for understanding the allelopathic mechanism about allelochemicals to plants.

Comprehensive evaluation of the allelopathic potential of Elymus nutans.

Elymus nutans has been widely planted together with other perennial grasses for rebuilding degraded alpine meadow atop the Qinghai-Tibetan Plateau. However, the rebuilt sown pastures begin to decline a few years after establishing. One of the possible causes for the degradation of sown grassland may come from allelopathy of planted grasses. The purpose of this study was to examine allelopathic potential of Elymus nutans. Three types of aqueous extract from Elymus nutans and its root zone soil were prepared, and 5 highland crops and 5 perennial grasses were used as recipient plants. Elymus nutans exhibited strong allelopathic potential on germination and seedling growth of 5 crops, but different crops or perennial grasses respond to the extract differently. The pieces aqueous extract have stronger inhibition than whole plant extract and root zone soil extract. Hordeum vulgar var. nudum, Avena sativa, and Festuca sinensis were the most affected, while Chenopodium quinoa and Elymus sibiricus were the least affected. Elymus nutans presented less influence on Poa pratensis and Poa crymophylla than on Festuca sinensis. It is recommended that the species combination of mixture for restoration should be considered for allopathic effects on the coseeding to decrease the seeding rate ratio of Elymus nutans. The annual dicot crop seeds of Chenopodium quinoa and Brassica napus can be used as alternative subsequent crop for the seed field of Elymus nutans monoculture.

Cloning and functional characterization of epidermis-specific promoter MtML1 from Medicago truncatula.

Epidermis-specific promoters are necessary for ectopic expression of specific functional genes such as the cuticle-related genes. Previous studies indicated that both ECERIFERUM 6 (AtCER6) and MERISTEM L1 LAYER (ATML1) promoters from Arabidopsis thaliana can drive gene expression specifically in the epidermis of shoot apical meristems (SAMs) and leaves. However, the epidermis-specific promoters from legume plants have not been reported. Here, we cloned a 5' flanking sequence from the upstream -2150 bp to the translational start ATG codon of MtML1 gene of legume model plant Medicago truncatula. PlantCARE analysis indicated that this sequence matches the characteristics of a promoter, having TATA box and CAAT box, as well as contains some conserved elements of epidermis-specific promoters like AtCER6 and ATML1 promoters. The ß-glucuronidase (GUS) histochemical analysis showed that MtML1 promoter can drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition. Furthermore, it can also control GUS expression in leaves and siliques rather than roots of the stably transformed Arabidopsis. More importantly, the leaf cross-section observations indicated that MtML1 exclusively expressed in the epidermis of leaves. These results suggested that MtML1 promoter performed the epidermis-specific in plant shoot. Our study establishes the foundation for driving the cuticle-related gene to express in epidermis, which may be very useful in genetic engineering of legume plants.