# Tissue-specificity of ROS signalling and production in Sarcocornia quinqueflora in the context of salinity stress tolerance

Ahmed Ibraheem Ahmed, H ORCID: 0000-0003-1990-8951 2021 , 'Tissue-specificity of ROS signalling and production in Sarcocornia quinqueflora in the context of salinity stress tolerance', PhD thesis, University of Tasmania.

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## Abstract

Soil salinity is predicted to become more severe and widespread adding more challenges for sustainable crops production worldwide. Salinity stress tolerance is a complex trait, both physiologically and genetically, and halophytes represent a rich resource for understanding different strategies used by plants to cope with saline condition. While many previous studies explored various physiological and genetic aspects of salinity tolerance in halophytes, most of them were focused on annual species. However, this knowledge is hardly applicable to perennial crops that need to maintain capability of growth and development for many successive seasons and, therefore, cannot rely on the strategy associated with salt exclusion from uptake. Succulence is one of that important strategies by which perennial halophytes conserve water and dilute immense salt concentration in their susceptible tissues. Succulent halophytes can be used as convenient models for understanding the mechanistic basis of plant adaptation to salt stress. This understanding is important for improving water use efficiency (WUE) in crop plants that undergo a salt-induced physiological drought under saline condition. How a perennial succulent halophyte then will manage this issue, morphologically, anatomically, and physiologically?
The major aim of this study was understanding the mechanistic basis of long-term salt tolerance strategy employed by the succulent perennial halophyte, Sarcocornia quinqueflora. The following specific objectives were addressed:
• To quantify the relative contribution of organic and inorganic osmolytes towards osmotic adjustment and turgor-induced growth in succulent shoots.
• To link osmotic adjustment and stomatal characteristics with salinity stress tolerance and water use efficiency (WUE) of succulent stems.
• To dissect specific morphological and anatomical features of succulent shoots and investigate their potential contribution toward their salt tolerance strategy.
• To investigate the causal relationship between salinity and oxidative stress tolerance in succulent stems of this perennial halophyte.
• To understand the role of ROS signalling in regulating activity of membrane transporters mediating ion homeostasis in this plant.
The whole-plant responses of S. quinqueflora to soil salinity was investigated using a broad range of salinity (0 – 1000 mM NaCl). S. quinqueflora showed the typical growth response of a succulent halophyte, with maximum growth obtained at 200 mM NaCl, while growth was reduced at concentrations exceeding 600 mM NaCl. Elevated salinity levels up to 400 mM NaCl largely promoted dry matter yield, succulence, shoot surface area and stomatal characteristics. The osmolality of shoot sap increased as salinity increased from 0 to 1000 mM NaCl. Osmotic adjustment in succulent shoot was achieved, even at the highest salinity levels, by a massive accumulation of inorganic ions, with Na$$^+$$ and Cl$$_-$$ contributing ~85 % of its osmolality, while organic compatible solutes and K$$^+$$ were responsible for only ~15%. These facts suggest that cell expansion growth in this species is relying heavily upon the coordination between the cell vacuolar sequestration capacity (VSC) of Na$$^+$$ and Cl$$_-$$ and the extent of the cell wall extensibility (CWE). The maximum VSC of Na$$^+$$ and Cl$$_-$$ is required to keep the cytosol toxic-free and to lower the cell osmotic potential which in turn elevates turgor pressure (hence, succulence). Carbohydrates were not reduced at high salinity compared to plants at optimal conditions, implying that growth retardation at severe salt dosages was attributed to limitations in VSC rather than inadequate photosynthesis and substrate limitation.
The control of stomatal operation seems to be critical for S. quinqueflora performance under saline conditions. The fact that transpiration rate was maintained unchanged over the very broad (200 to 1000 mM NaCl) range of salinities, despite the large difference in stomatal density and aperture size, suggest superior plant’s ability to optimize WUE and balance water loss with CO$$^2$$ assimilation. Importantly, shoot K$$^+$$ was unchanged across the entire range of salinity treatments (200 – 1000 mM NaCl) and positively correlated with transpiration rate (R=0.98), indicative of the likely role of K$$^+$$ in controlling stomatal transpiration. Therefore, the superior salt tolerance of succulent shoots is achieved by effective reliance on Na$$^+$$ and Cl$$_-$$ accumulation for osmoregulation and turgor-induced growth and maintaining K$$^+$$ threshold levels for efficient stomatal operation.
The leafy stems of S. quinqueflora shoot are composed of assimilating oblong internodes (beads), representing the main photosynthetic organ. Anatomically, the plant develops two distinct layers: an endodermis-like layer (ED), and an additional internal photosynthetic layer (IP). We followed the morpho-anatomical changes in S. quinqueflora leafy stem under varied salt levels in beads of different ages to assess this biological barrier (ED) from non-senescent to senescent stages. Our findings revealed that S. quinqueflora utilizes senescence process to discard excess salt being accumulated in outer tissues of their leafy stems (salt shedding). The development of ED and IP appears to be important to enable this process and determines the whole salt-coping strategy for the plant. Elevated salinity leads to an accelerated development of the ED. In addition, its development strongly affected ion distribution between outer (senescent) and inner (non-senescent) tissues. A positive correlation between the ratio of ED to a bead diameter and the outer to inner concentration of Na$$^+$$ was observed. These ratios were highest in older (basipetally-located) beads and progressively decreased towards the tip. The Na$$^+$$/K$$^+$$ ratio was substantially higher in outer region compared to the inner one. In addition, different leafy stem regions had showed that a ratio for any given NaCl concentration was the lowest for the top stem region with maximum impact being observed in the bottom stem region. High K$$^+$$ content in the tip of the leafy stem also drives the expansion growth of the plant. Accordingly, the top leafy stem region kept Na$$^+$$/K$$^+$$ ratio at the constant level regardless of external salt concentration. This correlates with the plant’s ability to grow/survival even at the highest (1000 mM) NaCl concentration tested. Furthermore, the Na$$^+$$/K$$^+$$ ratio in inner tissues of bottom beads at highest salinity treatments (800 and 1000 mM NaCl) that showed clear senescence symptoms was ~1.0, indicative of complete separation of the outer and inner tissues at late developmental stage due to the presence of the fully suberized endodermis multilayer (ED).
Accordingly, a model was suggested for bead tolerance strategy at early, middle, and late stages under saline conditions involving a suggested role for the internal photosynthetic layer (IP). Two main features are envisaged. The first one is an accelerated development of a biological barrier (ED) which, in its earlier developmental stage, control water and solute movements to the water storage tissue (WS) and determine Na$$^+$$/K$$^+$$ ratio in both senescing and non-senescing tissues. At a later stage, this tissue becomes multilayered and highly suberized, thus protecting the internal tissues for many successive seasons. The second feature is a difference in the energy supply (source) for the outer and the inner tissues. The outer layer (palisade tissue) will fuel the water storage cells to mediate Na$$^+$$ and Cl$$_-$$ sequestration in their vacuoles while the internal photosynthetic layer operates as an energy provider for young beads and roots.
The ability of S. quinqueflora to extend growth for many successive seasons under saline condition also implies an efficient and well-regulated ROS-scavenging and signalling systems at both organ and tissue levels. The causal relationship between activity of key membrane transporters involved in maintaining plant ionic homeostasis and oxidative stress tolerance in succulent stems was investigated. S. quinqueflora possess a well-developed antioxidant system, including betalains, ascorbic acid, α-tocopherol, polyphenols, and flavonoids. The optimal growth level (200 mM NaCl) had the lowest antioxidants concentrations, indicating its capability to maintain favorable ROS levels at moderate salinity. Moreover, both sugars and ROS were positively correlated suggesting that growth/biomass reduction at these conditions probably mediated by re-allocation of the energy pool towards sugar production to operate as non-enzymatic antioxidant scavengers. Also, a negative correlation was recorded between plant biomass and antioxidant activity, implying that the latter should be treated as a damage control mechanism rather than a trait that confers salinity tolerance.
In addition, ROS-induced net K$$^+$$ and Ca$$^{2+}$$ fluxes were measured from various bead tissues being located either in outer (palisade tissue, Pa; and water storage tissue, WS), or inner leafy stem part (internal photosynthetic layer, IP; and stele (vascular cylinder) parenchyma, SP) in addition to the in-between barrier (ED). Two types of ROS were used to induce ion fluxes, hydroxyl radical (OH$$^•$$) and hydrogen peroxide (H$$_2$$O$$_2$$). The flux responses to oxidative stresses were governed largely by (1) the type of ROS applied (OH$$^•$$ or H$$_2$$O$$_2$$); (2) the tissue-specific origin and function (parenchymatic or chlorenchymatic); and (3) the tissue location in either outer (senescent) or inner (non-senescent) bead part. ROS-induced K$$^+$$ effluxes that were highly tissue-specific, with inner tissues of the plant beads being more sensitive to ROS applied, as compared to the plant outer parts. The magnitude of ion flux response to OH$$^•$$ was higher compared to response to H$$_2$$O$$_2$$. The ability to retain cellular K$$^+$$ under OH$$^•$$ stress varied between different bead tissues and was ranked in the following descending order: WS > Pa > IP > SP. Hydroxyl radicals (OH$$^•$$) always led to Ca$$^{2+}$$ influx with all bead tissues, while treatment with H$$_2$$O$$_2$$ induced opposite Ca$$^{2+}$$ flux responses: the Ca$$^{2+}$$ influx was recorded only from the photosynthetically active tissues (Pa and IP), while the parenchyma tissues (WS and SP) had transient Ca$$^{2+}$$ efflux. These results indicate high tissue-specific ability of S. quinqueflora to maintain their ion homeostasis upon exposure to ROS; a feature that is mainly determined by the developed suberized barrier (ED).
In conclusion, the salt tolerance strategy of perennial S. quinqueflora halophyte relies on a set of morphological, anatomical, and physiological traits, enabling the ability to extend growth for many successive seasons. This work emphasized the importance and a requirement to engage specific anatomical features in studying salinity tolerance mechanisms of halophytes, in addition to the traditional whole-plant phenotyping. To the best of our knowledge, none of the previous works provided a causal link between salinity-stress tolerance and ROS activation of ion transporters mediating ionic homeostasis in S. quinqueflora succulent tissues. This gap in our knowledge was filled by the current study. The future work should be focused on comparing tissue-specificity of long-term salinity tolerance mechanisms in other perennial succulent and non-succulent halophytes.

Item Type: Thesis - PhD Ahmed Ibraheem Ahmed, H reactive oxygen species; salinity stress; ion flux; succulence; tissue-specificity; perennial halophytes; anatomical adaptations; water-use-efficiency Copyright 2021 the author View statistics for this item