Dynamic responses of physiology, biochemistry and structure of vegetative organs of Juglans nigra to salt stress

Abstract: 【Objective】This study aimed to investigate the physiological and biological responses and anatomical stracture changes of Juglans nigra vegetative organs to persistent salt stress in order to understand the adaptation mechanism of J. nigra to salt stress.【Methods】Seedlings of J. nigra were used as materials, and a 42-day pot experiment was conducted with four salt concentration gradients (0 mmol·L^-1 , 50 mmol·L^-1 , 100 mmol·L^-1 , and 200 mmol·L^-1 ). Functional leaves, stems, and first lateral roots were collected at 14, 28, and 42 days after salt stress for the determination of physiological and biochemical indicators as well as anatomical structure of the vegetative organs of the seedlings. The dynamic changes in physiology, biochemistry and anatomical structure of the seedlings were studied, and the relationship between physiological, biochemical and anatomical parameters of the vegetative organs was analyzed. Principal component analysis (PCA) was used to determine the salt tolerance evaluation index of J. nigra.【Results】The relative water content (RWC) decreased with increasing salt concentration，reaching 56.15% under 200 mmol·L^{- 1}  treatment on the 42nd day of salt stress. The malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, ascorbate peroxidase (APX) activity, and total flavonoid compounds (TPC) all significantly increased under salt stress. On the 14th day of stress,with the increase in salt concentration, the total phenolic content (TFC) and proline (Pro) content initially decreased then increased, and the total phenolic content (TFC) and proline (Pro) content increased significantly under salt treatment at the 28th and 42nd day of stress. With the increase in salt concentration, the soluble protein (SP) content showed a trend of initial increase and subsequent decrease at the 14th day of stress, and increased at the 28th day, and showed a trend of initial decrease and subsequent increase at the 42th day; There was no significant difference between the content of soluble sugar (SS) under salt treatment and the control at the 14th day. With the increase of salt concentration, the content of soluble sugar (SS) on the 28th day increased, and the content of soluble sugar (SS) on the 42nd day increased first, then decreased and then increased. The palisade tissue thickness (PT) increased at first and then decreased with the increase of salt concentration, and was increased significantly by treatment at 50 mmol·L^-1 , and decreased significantly by treatments at 100 mmol·L^-1 and 200 mmol·L^-1 as compared with the control. The spongy tissue thickness (ST) and leaf lamina thickness (LT) increased compared with the control on the 14th and 28th day under salt stress, but decreased on the 42nd day. The thickness of the upper epidermis (UE) under salt treatment increased on the 14th day of stress, increased at first and then decreased on the 28th day after stress, increased significantly at 50 mmol·L^{- 1} , and decreased significantly at 200 mmol·L^-1 , which was 26.06% lower than the control. With the increase of salt concentration, the thickness of the lower epidermis (LE) decreased first and then increased on the 14th day of salt stress, increased first and then decreased on the 28th day, and decreased on the 42nd day. With the increase of salt concentration, the cell tightness ratio (CTR) and the ratio of palisade tissue to spongy tissue (PT/ST) increased first and then decreased on the 14th and 42nd day, and decreased on the 28th day. The cell porosity ratio (SR) increased with the increase of salt concentration on the 14th and 28th day. Subsequently it decreased at first and then increased on the 42nd day, reaching the minimum value of 39.87% at 100 mmol·L-1 . As the salt concentration increases, the thickness of vascular bundle (VBT) of leaves decreased on the 14th day. It increased at first and then decreased on the 28th day, and decreased significantly at 100 mmol · L- 1 and 200 mmol · L- 1 on the 42nd day. Leaf main vein diameter (MVT) and midrib protuberant degree (MPD) of leaves under salt stress decreased significantly on the 14th day, increased first and then decreased with the increase of salt concentration on the 28th day, and decreased on the 42nd day. The thickness of the stem xylem tissue (SXT) significantly increased under salt treatment, and reached the maximum value of 522.88 μm under 200 mmol·L^-1 treatment at day 42. The steam xylem thickness (SXT) significantly increased under salt treatment, and reached the maximum value of 522.88 μm under 200 mmol·L^{- 1} treatment on the 42nd day. As the salt concentration increases, the stem phloem thickness (SPhT) increased on the 14th day, increased first and then decreased on the 28th day, and decreased significantly on the 42nd day at 100 and 200 mmol·L^-1 . The stem cortical thickness (SCT) significantly increased under salt treatment, reaching a maximum value of 327.84 μm on the 42nd day under 200 mmol·L^-1 treatment. With the increase of salt concentration, the stem periderm thickness (SPT) increased first and then decreased. Root diameter (RD) and root cortical thickness (RCT) increased at first and then decreased on the 14th and 28th day, and they decreased significantly at 50 mmol·L^-1 and 200 mmol·L^-1 on the 42nd day. As the salt concentration increases, root vascular bundle diameter (RVBT) decreased on the 14th day, increased on the 28th day, and decreased significantly on the 42nd day. With the increase of salt concentration, the root periderm thickness (RPT) increased first and then decreased on the 14th day, decreased on the 28th day, and decreased first and then increased on the 42nd day. The main components of root, stem and leaf under different salt concentrations were significantly different in the three stress periods. RD, Pt/St, RCT,SPT, and CTR were the first principal components on the 14th day of stress, while PT/St, SCT, RPT, SX T, SPT were the first principal components on the 28th day of stress, the first principal components of stress on the 42nd day were VBT, SPT, SXT, MVT, and SPhT. The physiological and biochemical indexes were correlated with the anatomical characteristics, and the anatomical structure of the stem was strongly correlated with RWC, anti-reactive oxygen species (SOD、APX、TPC、TFC) and osmotic regulators (Pro、SP、SS). TPC was closely related to mesophyll, vein and root diameter. SPT had strong correlation with leaf veins.【Conclusion】J. nigra responded to salt stress by altering the structures of vegetative organs, increasing osmoregulatory substances, and enhancing antioxidants. Seedlings possess a certain degree of salt tolerance ability when exposed to salt concentrations below 50 mmol·L^-1 . PT/ST, SCT, SXT, and RD can serve as reliable indicators for screening salt-tolerant varieties in seedlings.