Physico-biochemical and molecular responses of Acacia auriculiformis to salinity stress

Salinity limits the growth and yield of many crops across the globe and is considered as major threat to agriculture. Acacia auriculiformis, an important salt tolerant crop, is growing abundantly in the salt-affected mangrove areas of Sunderban, West Bengal, India. In the present study, we have reported the physiological, molecular and antioxidant response of this crop to a gradient of salt treatments ranging between 0 and 800 mM NaCl. As a stress response, the antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) were highly activated at 200, 400 and 800 mM NaCl respectively. Antioxidant metabolites such as phenols and thiols elevated with increasing salt treatments thus augmenting antioxidant activity with significant positive relationship with phenol content. Similarly, phenylalanine ammonia lyase (PAL) activity was up-regulated in a dose dependent manner with significant relationship with phenol content. This study also reported the phenolic profile for the first time in A. auriculiformis with the abundance of flavonoids. In addition, transcriptional up-regulation of Na+/H+ antiporter gene (NHX1) and the development of robust vascular tissues was noticed at 400 mM NaCl stress compared to control, while further stress at 800 mM NaCl induced poor vascular tissue growth but with higher PAL activity and consequent higher phenol content. Based on this observation, a model for salt tolerance mechanism of A. auriculiformis has been proposed.


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Salinity is known to severely affect the growth and yield of many crops in arid and semi-arid

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Acacia auriculiformis, a representative member of Fabaceae, is reputed for its use as fodder 62 and textile industry because of its high tannin content. Moreover, this crop has the potential to 63 find its place in social forestry and cropland agroforestry because of its fast growing nature 64 and wide adaptability to diverse environmental conditions [15]. In West Bengal, India, this 65 crop is grown abundantly in costal salt-affected region of Sunderban and exhibits a high 66 degree of salt tolerance. The underlying salt tolerance mechanism of this crop is reported to be 67 closely associated with ion selectivity, increased accumulation of osmoprotectants, 68 sequesteration of Na in root tissues, anatomical adjustments, Na exclusion and tissue tolerance 4 69 mechanism [14,16]. However, antioxidant metabolism in response to salinity stress in A. 70 auriculiformis is ambiguous and not well documented.

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Therefore, the present study was extended to examine the activities of antioxidant enzymes viz. 72 SOD, CAT, GPX and PAL in response to a gradient of salinity stress, ranging from 0 to 800 73 mM NaCl. Moreover, total phenol content with its profile of phenolic compounds and 74 associated antioxidant activity were also explored. Salt induced physiological changes such as 75 cross-linking of cell wall components and vascular tissue development through histological 76 staining have also been noticed. Additionally, gene expression of Na + /H + -antiportes in A. 77 auriculiformis at 400 mM has been reported.

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Salinity treatment and morphological study 80 The three month-old seedlings of A. auriculiformis, collected from salt affected areas of 81 Sunderban, South 24 Parganas, West Bengal, India and grown in sandy-clay soil in pots (25 cm 82 diameter × 20 cm height) in three replicates at the University green house at Mohanpur, Nadia, 83 West Bengal and treated each with 0, 200, 400, 600 and 800 mM Nacl for consecutive 10 days.

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The growth pattern was analyzed. For biochemical studies, the leaves from each treatment 85 replications were harvested and preserved at (-) 80 °C until further use. the photochemical NBT method in terms of SOD's ability to inhibit reduction of NBT to form 92 formazon by superoxide. The photoreduction of NBT was measured at 560 nm. The SOD 93 activity was expressed in units, which were defined as amount of sample required to cause 50% 94 inhibition of photo-reduction of NBT per minute. CAT (EC 1.11.1.6) and GPX (EC 1.11.1.7) 95 were extracted in 100 mM phosphate buffer (pH 7.5) containing 2% PVP and 0.25 % Triton-X.   NaCl, but the stimulatory effect was more pronounced at 400 mM. On the contrary, at 600 and 157 800 mM salt treatment, SOD activity declined significantly below control. Hence, the SOD 158 activity might be either up-regulated or down-regulated depending on the severity of stress. The 159 result however, suggested that relatively higher H 2 O 2 generation was imposed at lower (200 and 8 160 400 mM) than higher (600 and 800 mM) salt concentration. The CAT activity (Fig. 1B), on the 161 other hand, increased significantly over control with all the treatments except 400 mM. It was 162 further noticed that PAL (Fig. 1C) activity and GPX activity ( Fig. 2A), the key enzymes of 163 phenol synthesis and oxidation respectively, controlling the phenol accumulation in plants, 164 increased significantly over control in response to increasing salt treatments.

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Effect of salt treatment on content of phenolic compounds and reduced glutathione: 166 Total phenol content (Fig. 2B) increased significantly over control with increasing level of salt 167 treatments. Similar to phenol, total thiol content increased significantly with increasing salinity 168 (Fig. 2C). In the present study an attempt has been made for analysis of 16 phenolic compounds  Salt treatment with 200 and 400mM NaCl did not produce any significant differences in gallic 180 acid content with the control. However, gallic acid content at 600 and 800 mM has found to 181 differ significantly not only between them but also from other treatments. Protocatechuic acid 182 with its share of 6.28% formed the third major phenolic compound and increased significantly at The antioxidant activity, measured under DPPH and FRAP assay (Fig. 4), was significantly

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Differential salt stress depicted anatomical changes in A. auriculiformis (Fig. 5B). 205 Pholoroglucinol staining documented significant changes in vascular tissues across different 10 206 treatments. Pholoroglucinol staining established more cross-linking of cell wall components with 207 400 mM NaCl but less with 800 mM NaCl treated phyllode over control. In addition, 400 mM 208 NaCl stressed phyllode exhibited better vascular tissue development as opposed to 800 mM 209 NaCl stressed condition and even better over control. Additionally, the phloem tissue 210 development in the phallode was found to increase in the order of 800 mM < control < 400 mM 211 NaCl treated seedlings. As 400 mM NaCl treatment depicted more cross-linking in seedling 212 compared to control as well as 800 mM NaCl treatment, the transcript expression of a Na + /H + 213 antiporters gene (NHX1) was analyzed in 400 mM NaCl treated seedlings. Relative transcript 214 abundance of NHX1 gene was found to be up regulated at 400 mM NaCl stress compared to 215 untreated control plants (Fig. 5C). Quantification of relative expression of Na + /H + antiporters 216 (relative to Actin) conducted through imajej software analysis revealed that upon 400 mM NaCl 217 stress the gene was 2.84 fold up-regulated compared to control plants (Fig. 5D).

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In the present study, decline in number of leaves observed with increasing salinity suggested that 223 the growth reduction appears to be related to impairment of either non-cyclic photosynthetic 224 electron transport or CO 2 assimilation. Earlier study reported that the growth related parameters 225 including stomatal conductance, photosynthetic pigments, protein and carbohydrate content in A.