Increasing soil salinity reduces crop yields worldwide, with rice being particularly affected. We have examined the correlation between apoplastic barrier formation in roots, Na+ uptake into shoots and plant survival for three rice (Oryza sativa L.) cultivars of varying salt sensitivity: the salt-tolerant Pokkali, moderately tolerant Jaya and sensitive IR20. Rice plants grown hydroponically or in soil for 1 month were subjected to both severe and moderate salinity stress. Apoplastic barriers in roots were visualized using fluorescence microscopy and their chemical composition determined by gas chromatography and mass spectrometry. Na+ content was estimated by flame photometry. Suberization of apoplastic barriers in roots of Pokkali was the most extensive of the three cultivars, while Na+ accumulation in the shoots was the least. Saline stress induced the strengthening of these barriers in both sensitive and tolerant cultivars, with increase in mRNAs encoding suberin biosynthetic enzymes being detectable within 30 min of stress. Enhanced barriers were detected after several days of moderate stress. Overall, more extensive apoplastic barriers in roots correlated with reduced Na+ uptake and enhanced survival when challenged with high salinity.

Rice is an important crop that is very sensitive to salinity. However, some varieties differ greatly in this feature, making investigations of salinity tolerance mechanisms possible. The cultivar Pokkali is salinity tolerant and is known to have more extensive hydrophobic barriers in its roots than does IR20, a more sensitive cultivar. These barriers located in the root endodermis and exodermis prevent the direct entry of external fluid into the stele. However, it is known that in the case of rice, these barriers are bypassed by most of the Na+ that enters the shoot. Exposing plants to a moderate stress of 100 mM NaCl resulted in deposition of additional hydrophobic aliphatic suberin in both cultivars. The present study demonstrated that Pokkali roots have a lower permeability to water (measured using a pressure chamber) than those of IR20. Conditioning plants with 100 mM NaCl effectively reduced Na+ accumulation in the shoot and improved survival of the plants when they were subsequently subjected to a lethal stress of 200 mM NaCl. The Na+ accumulated during the conditioning period was rapidly released when the plants were returned to the control medium. It has been suggested that the location of the bypass flow is around young lateral roots, the early development of which disrupts the continuity of the endodermal and exodermal Casparian bands. However, in the present study, the observed increase in lateral root densities during stress in both cultivars did not correlate with bypass flow. Overall the data suggest that in rice roots Na+ bypass flow is reduced by the deposition of apoplastic barriers, leading to improved plant survival under salt stress.

Soil salinity is a major factor affecting crop productivity worldwide. This study explores mechanisms that contribute to salt tolerance in rice (Oryza sativa L.). Hydroponically grown, 2‐week‐old salt tolerant and sensitive indica rice varieties, Pokkali and Jaya, respectively, were exposed to a 48‐h stress period with NaCl (0–250 mM). When exposed to 200 mM NaCl, micromolar levels of external Ca2+ elevated survival of both varieties. The Ca2+ levels required were lower for Pokkali than for Jaya, but resulted in significantly higher survival. Estimates of Na+ and K+ in root and shoot compartments were made by flame photometry, while X‐ray microanalysis was used to localize Na+ in the extracellular matrix of the shoot. Transpirational bypass flow was estimated using the apoplastic tracer, 8‐hydroxypyrene‐1,3,6‐trisulphonic acid, trisodium salt. Our data demonstrate a Ca2+‐dependent reduction in Na+ transport to shoots, which correlated with a decline in bypass flow and of Na+ in the transpirational stream. In addition, the Na+ that enters the shoot is partitioned among several distinct compartments. Survival is inversely correlated with Na+ levels in the shoot apoplastic fluid, which surrounds the cell and influences cytosolic composition. Pokkali maintained lower Na+ in its apoplast compared with the salt sensitive Jaya at the same total shoot Na+. Na+ in the apoplast appears to be regulated by sequestration into intracellular compartments. This sink supplements the primary response of reducing Na+ influx into the shoot and effectively buffers the apoplastic fluid in Pokkali. All of these mechanisms are operational in Jaya as well but are deployed less effectively.

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell-wall barriers to thrive in saline conditions. We studied a Cytochrome P450 gene from A. officinalis, AoCYP94B1, and its putative ortholog in Arabidopsis (Arabidopsis thaliana), AtCYP94B1, which are involved in apoplastic barrier formation. Both genes were induced by 30 minutes of salt treatment in the roots. Heterologous expression of AoCYP94B1 in the atcyp94b1 Arabidopsis mutant and wild-type rice (Oryza sativa) conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and GC-MS/MS quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation, yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt-sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in the atwrky33 background. This further confirmed that AtWRKY33-mediated regulation of AtCYP94B1 is part of the salt tolerance mechanism. Our findings may help efforts aimed at generating salt-tolerant crops.

Salinity affects growth and development of plants, but mangroves exhibit exceptional salt tolerance. With direct exposure to salinity, mangrove roots possess specific adaptations to tolerate salt stress. Therefore, studying the early effects of salt on mangrove roots can help us better understand the tolerance mechanisms. Using two-month-old greenhouse-grown seedlings of the mangrove tree Avicennia officinalis subjected to NaCl treatment, we profiled gene expression changes in the roots by RNA-sequencing. Of the 6547 genes that were differentially regulated in response to salt treatment, 1404 and 5213 genes were significantly up- and down-regulated, respectively. By comparative genomics, 93 key salt tolerance-related genes were identified of which 47 were up-regulated. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in ethylene and auxin signaling were up-regulated while those involved in ABA signaling were down-regulated. These results imply that ABA-independent signaling pathways also play a major role in salt tolerance of A. officinalis. Further, ethylene response factors (ERFs) were abundantly expressed upon salt treatment and the Arabidopsis mutant aterf115, a homolog of AoERF114 is characterized. Overall, our results would help in understanding the possible molecular mechanism underlying salt tolerance in plants.

Salt exclusion at the roots and salt secretion in the leaves were examined in a mangrove, Avicennia officinalis. The nonsecretor mangrove Bruguiera cylindrica was used for comparative study of hydrophobic barrier formation in the roots. Bypass flow was reduced when seedlings were previously treated with high salt concentration. A biseriate exodermis was detected in the salt-treated roots, along with an enhanced deposition of hydrophobic barriers in the endodermis. These barriers reduced Na + loading into the xylem, accounting for a 90-95% salt exclusion in A. officinalis. Prominent barriers were found in the roots of B. cylindrica even in the absence of salt treatment. A cytochrome P450 gene that may regulate suberin biosynthesis was up-regulated within hours of salt treatment in A. officinalis roots and leaves, corresponding with increased suberin deposition. X-ray microanalysis showed preferential deposition of Na + and Cl − in the root cortex compared with the stele, suggesting that the endodermis is the primary site of salt exclusion. Enhanced salt secretion and increased suberin deposition surrounding the salt glands were seen in the leaves with salt treatment. Overall, these data show that the deposition of apoplastic barriers increases resistance to bypass flow leading to efficient salt exclusion at the roots in mangroves.

Class I TREHALOSE-PHOSPHATE-SYNTHASE (TPS) genes affect salinity tolerance and plant development. However, the function of class II TPS genes and their underlying mechanisms of action are unknown.We report the identification and functional analysis of a rice class II TPS gene (OsTPS8). The ostps8 mutant was characterised by GC-MS analysis, an abscisic acid (ABA) sensitivity test and by generating transgenic lines. To identify the underlying mechanism, gene expression analyses, genetic complementation and examination of suberin deposition in the roots were conducted.The ostps8 mutant showed salt sensitivity, ABA sensitivity and altered agronomic traits compared to the wild-type (WT), which could be rescued upon complementation. The dsRNAi line phenocopied the mutant, while the overexpression lines exhibited enhanced salt tolerance. The ostps8 mutant showed significantly reduced soluble sugars, Casparian bands and suberin deposition in the roots compared to the WT and overexpression lines. The mutant also showed downregulation of SAPKs (rice SnRK2s) and ABA-responsive genes. Furthermore, ostps8pUBI::SAPK9 rescued the salt-sensitive phenotype of ostps8.Our results suggest that OsTPS8 may regulate suberin deposition in rice through ABA signalling. Additionally, SAPK9-mediated regulation of altered ABA-responsive genes helps to confer salinity tolerance. Overexpression of OsTPS8 was adequate to confer enhanced salinity tolerance without any yield penalty, suggesting its usefulness in rice genetic improvement.

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