Photosynthesis, ionic relations, and Ca<sup>2+</sup> signaling in leaves of glycophytic <i>Oryza sativa</i> L. and its wild relative halophytic <i>Oryza coarctata</i> L. in the context of differential salinity stress tolerance
Creators: name: Yun, Ping
Publisher: name: University of Tasmania
Year: 2024

description: Salinized soil (EC &gt; 4 dS/m) is a worldwide environmental problem that affects crop growth and yield, threatening global food security. Because of the expansion of salt?affected soil area, urbanization extension-caused shrink of arable land, and increased food demand due to growing population, exploiting marginal lands, including saline soil, for food production seems to be inevitable. However, most crops are glycophytes and salinity significantly suppresses their production. As the second most widely used grain crop, rice (<i>Oryza sativa</i>) feeds half of the world’s population. Its threshold of salt tolerance, on the other hand, is very low (3.5 dS/m causes 50% yield loss). To improve salt tolerance in cultivated rice (<i>O. sativa)</i>, an adequate way is to reintroduce the traits employed by its salt-tolerant wild relatives. As the only halophyte in the <i>Oryza </i>species, O. coarctata therefore represents a valuable resource for achieving this goal.<br>Plant yield is ultimately proportional to its photosynthetic capacity; however, the molecular basis of salinity tissue tolerance in leaf mesophyll remains largely unexplored, as compared with plant roots. The aim of this study was to better understand the mechanistic basis of salt tolerance in the leaf of <i>O. coarctata</i> and identify some potential target traits for the breeding program in rice salt tolerance enhancement. This was achieved by a comparative analysis between salt-sensitive cultivated rice <i>Oryza sativa</i> (cv. Koshihikari) and its wild relative <i>O. coarctata</i>.<br>Firstly, we have compared gas exchange characteristics and leaf photochemistry (Chapter 4). Our results showed that the developed stomata in cultivated rice were smaller (stomatal aperture reduced by 23%) and denser (density increased by 22%) under saline conditions (100 mM NaCl treated for 4 weeks) which resulted in a 68% smaller stomatal conductance (Gs) and a 70% lower maximum stomatal opening speed. Two parameters in the Calvin cycle, the maximum rates of RuBisCO carboxylation (V<sub>cmax</sub>) and electron transport through photosystem II for regeneration of RuBP (J<sub>max</sub>), were also considerably reduced by up to 51% in cultivated rice in response to salinity. In addition, excessive accumulation of Na+ in leaves of <i>O. sativa</i> led to a significant decrease in PSⅡ activity (chlorophyll fluorescence Fv/Fm ratio). Taken together, these changes resulted in a 43% decrease in the CO<sub>2</sub> assimilation rate in cultivated rice. On the contrary, <i>O. coarctata</i> possesses much larger intrinsic stomata, and the stomatal patterns (size, density, and opening speed) are unaffected by salt treatment. Salinity also induced V<sub>cmax</sub> and J<sub>max </sub>in wild rice. Moreover, the leaf Na+ content in <i>O. coarctata</i> remained at low levels after being exposed to NaCl. These features allowed <i>O. coarctata</i> to maintain relatively steady CO2 assimilation under salt conditions.<br>Since <i>O</i><i>. coarctata</i> showed a better ability than <i>O. sativa</i> to sustain a relatively stable leaf metabolism process in response to salinity, we then conducted experiments on leaf mesophyll cells from the ionic perspective (ion flux, content, transporter/channel expression, etc.) to examine the leaf salinity tissue tolerance in both species (Chapter 5). Salt treatment induced a 15% decline in cell viability of leaf mesophyll cells in <i>O. coarctata,</i> while the reduction in <i>O. sativa</i> was ca 80%. In addition, <i>O. coarctata</i> had increased K<sup>+</sup> content, less Na<sup>+</sup> amount, and higher Cl- level compared to its cultivated counterpart under NaCl conditions. Electrophysiological experiments showed that wild rice possessed better K<sup>+</sup> retention ability (less K<sup>+</sup> efflux) and much less Na<sup>+</sup> uptake than cultivated rice when responding to transient NaCl treatment. Pharmacological experiments indicated that non-selective cation channels (NSCCs) were involved in the Na<sup>+</sup> influx and K<sup>+</sup> efflux in leaf mesophyll cells, while salt-induced K<sup>+</sup> loss in <i>O. sativa</i> was also mediated by the guard cell outward-rectifying K<sup>+</sup> channel (GORK). The low Na+ contents in <i>O. coarctata</i> was also contributed by salt secretion via leaf microhairs (Chapter 7). In addition, salinity triggered depolarization of plasma membrane (PM) in mesophyll cells of <i>O. sativa </i>which is most likely because of massive Na<sup>+</sup> accumulation in leaf blades. In contrast,<i> O. coarctata</i> demonstrated a NaCl dose-dependent PM hyperpolarization in mesophyll cells due to the preference for Cl- uptake. Transcriptional analysis suggests that several transporters (AKT1, SOS1, HKT1, and CLC1) may contribute to the difference in ionic relations between <i>O. coarctata</i> and <i>O. sativa.</i><br>ROS and Ca<sup>2+</sup> signaling are involved in salinity stress response and regulation of ion channels/transporters. As <i>O</i>. <i>coarctata</i> demonstrated a remarkable salt tolerance in leaf tissue and its outstanding capability in maintaining ionic homeostasis, we hypothesized that this process could be mediated by ROS and Ca<sup>2+</sup> signaling. Hence, a series of experiments were carried out, including the salt-induced ROS and antioxidant levels determination, ion fluxes (K<sup>+</sup>, Ca<sup>2+</sup>, and Na<sup>+</sup>) measurements in response to ROS treatment, and expression of ROS-producing and Ca<sup>2+</sup> signaling-related genes (Chapter 6). The results showed that <i>O. coarctata</i> had lower leaf overall ROS level and H<sub>2</sub>O<sub>2</sub> content than <i>O. sativa</i> under salinity conditions, which was likely due to the high levels of non-enzymatic antioxidants (total phenols, ascorbic acid, and α-tocopherol). The salt<i>-</i>induced ROS generation in both species was attributed to the NADPH oxidases, while the transcription level of its coded gene<i> RBOH</i> in <i>O.</i> <i>coarctata</i> was higher. Wild rice possessed a stronger Ca<sup>2+</sup> signal (73% higher Ca<sup>2+</sup> influx) and lower K<sup>+</sup> efflux (62%) than cultivated rice in response to H<sub>2</sub>O<sub>2</sub> treatment. In addition, the expressions of Ca2+ signaling related <i>CBL-CIPK</i> genes were rapidly upregulated (less than 1 h) by NaCl treatment in both species, whereas their extent in <i>O. coarctata</i> is greater<i>.</i><br>Collectively, <i>O. coarctata</i> possesses a superior salinity tolerance in leaf tissue and extraordinary capability to maintain cellular ionic homeostasis including K<sup>+</sup> retention, Na<sup>+</sup> exclusion, and Cl- utilization, which is partially attributed to the ROS-induced Ca<sup>2+</sup> signaling-related CBL-CIPK pathway. On top of that, the abundant non-enzymatic antioxidants in <i>O. coarctata</i> prevent the overaccumulation of ROS under saline conditions. Together with salt secretion, better leaf photochemistry, unaltered stomatal traits, and enhanced RuBisCO carboxylation and RuBP regeneration in the Calvin cycle, <i>O. coarctata</i> demonstrates a relatively steady photosynthetic rate and better growth under salinity conditions. Hence, these traits may be targeted in the rice breeding program for salt tolerance improvement<i>.</i>