| The freezing injury limited the distribution of plants and the chilling stress is one of the natural disasters prevailed in China. The huge economic losses resulted from freezing injury were concerned seriously by Chinese Government and scientific researchers. Therefore, studies on adaptive mechanisms of plants to chilling stress are very important;it will not only be a guide to introduction and cultivation of plants but also help to breed anti-freezing plants. However, there was no report about the physiological mechanism of evergreen woody plants of Sabina in northwest China during winter stress. The leaves of Sabina przewalskii and Sabina chinensis were used to explore the cold resistant mechanism of evergreen woody plants of Sabina by examining seasonal changes of the endogenous nitric oxide, anti-oxidative system, water content, osmoregulation substance, element, microstructure, ultra structure, stable carbon isotope. The results showed as follows:(1) The activities of CAT, POD and APX, contents of GSH, Pro and Car increased with temperature decrease, and the highest value occurred in the winter. The MDA content, NO release rate, NOS and NR activities increased markedly and reached two peaks in autumn and early spring, respectively. Meanwhile, the activities of POD, CAT, APX and the contents of GSH, Car and Pro were negatively correlated with mean temperature and MDA content in both species, it indicated that the anti-oxidative systems played an important role in potentiating freezing-resistance of plant and limiting the production of free radicals to protect membrane integrity. Furthermore, NO generation is always found before increasing of anti-oxidative enzyme activity and antioxidant content, it indicated that NO as an early signal molecule is able to induce the freezing tolerance by activating the anti-oxidative system involved in both anti-oxidative enzyme and antioxidant. S. przewalskii showed a prevailing strategy in signal function of NO and the higher anti-oxidative enzyme activity and antioxidant content than S. chinensis in order to develop the tolerance to freezing.(2) In the leaves of the two species, the relative water content (RWC) and free water content (FWC) decreased during hardening in autumn, but bound water content (BWC)and organic osmoticas accumulated and remained relatively higher level over winter. In spring, RWC and FWC increased, BWC decreased, and organic osmotica accumulated again. These seasonal patterns were consistent with winter hardening and dehardening. Organic osmotica may supply nutrients to regrowth in spring and contribute to the stabilization of the cell membrane by preventing lipid peroxidation and by binding the tissue water. The results also suggested that a better capacity for osmoregulation in S. przewalskii might account for its greater capacity for freezing tolerance than S. chinensis.(3) The concentrations of N, K and Cl in the leaves of two species in summer and autumn were higher than that in winter and spring, and this indicated that two species enriched abundant nutrient matters in summer and autumn, which were identical with accumulating higher biomass in these two seasons. The concentrations of P, Ca, Mg, Na, Si and S in the leaves of two species in winter and spring were higher than that in summer and autumn, and this indicated that two species could accumulate abundant inorganic osmotica to adapt itself to low temperature by increasing osmotic pressure of the cell. The results also suggested that a better capacity for accumulating inorganic osmotica in S. przewalskii might account for its greater capacity for freezing tolerance than S. chinensis.(4) It was shown that the foliar surface cells were covered with thick cuticular membrane, and the well-developed arenchyma appeared inside of their mesophyll in S. przewalskii and S. chinensis. It was observed in the two species that starch grains in chloroplasts accumulated in growing season and disappeared or diminished as soluble sugar content increased in cold season, but there was a larger increase in soluble sugar content in S. przewalskii than S. chinensis. Chloroplasts of S. chinensis were injured in winter, with some chloroplasts become abnormal, plastoglobuli increased and some lipid drops were observed, but no obvious injury in the chloroplasts of S. przewalskii had been observed. Mitochondria of both species remained stable and intact in dormancy season. It can be concluded that cold tolerance of two species related to the well-developed arenchyma, accumulation of starch grains in developing seasons, and increase of soluble sugar in dormancy season. Moreover, more stable chloroplasts and more marked increase of soluble sugar content in S. przewalskii under low-temperature stress might account for its greater capacity for freezing tolerance than S. chinensis.(5) The 5BC values in the leaves of the two species declined with monthly average temperature decreasing and maintained a relatively lower value in the whole winter. It indicated that the two species consumed more self substances in order to adapt to low temperature. As a result, the 813C values decreased. The 813C values of S. przewalskii were markedly higher than those of S. chinensis, it suggested that S. chinensis must consume more self substances than S. przewalskii under low-temperature stress. S. przewalskii showed a more prevailing strategy than & chinensis in order to develop the tolerance to freezing. Furthermore, very significant linear relationships were observed between 613C value and monthly average temperature, between 513C value and Pro content, and between 513C value and RWC in the leaves of S. przewalskii and S. chinensis. These results suggested that the 813C values of two species of Sabina can be used as another index to evaluate freezing tolerance.In conclusion, the well-developed arenchyma, accumulation of starch grains in growing seasons, increase of soluble sugar in dormancy season, signal function of NO to activate the anti-oxidative system, accumulation of organic and inorganic osmotica, stability of chloroplasts, and decrease of 513C value under low-temperature stress were important adaptive mechanisms of plants of Sabina in order to develop the tolerance to freezing.
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