Introduction

Exposure to prolonged salinity causes a decline in the growth and photosynthesis of plants1. Salinity is considered a non-optimal and non-invasive indicator for plant growth and photosynthesis2. Plant growth and net photosynthesis are interlinked and depend on carbon use efficiency3. Salt stress reduces CO2 assimilation due to stomatal limitation, leading to a decline in photosynthesis and blocking the activity of the Rubisco enzyme4,5,6. A decline in CO2 in stresses maintains water use efficiency (WUE) and the rate of transpiration for survival7. Stomatal closure reduces the expenditure of ATP and NADPH to maintain the temperature of the cell8. This response to salt intensity and duration depends on the type of plant species9,10,11. Higher intensities and long-term exposure in salt-sensitive species alter physio-biochemical processes and cease oxygenase and carboxylase enzymes. This also limits the formation of 1,5-bisphosphate (RuBP) triose phosphate utilization and hinders cyclic electron flow during phosphorylation12.

The halophytic plant species employ various strategies to counteract salt stress. These species accumulate a substantial amount of photosynthetic pigments. They also show enhanced photosynthesis, maintenance of cell osmotica by an accumulation of compatible osmolytes, effectively maintaining water use efficiency, and reducing the transpiration rate by stomatal regulations to thrive under salinity-induced stress13,14. Higher salt extremes and prolonged durations favor efficient growth to complete their life cycle15. These plants adapted to a multitude of modifications to cope with salt by changing the structural and functional facets16. Principal biochemical and physiological strategies to counteract salt extremities include the accumulation of organic osmolytes to scavenge ROS and free radicles, exclusion of toxic ions and compartmentalization, Na+/Cl sequestrations in roots, and accumulation of plenty of K+ and Ca2+ in leaves17,18. Salt-tolerant plants intensify cellular antioxidants to reduce lipid peroxidation (LPX) to maintain the membrane integrity and levels of reactive oxygen species (ROS) to prevent oxidative damage19. Accumulating compatible osmolytes in halophytes helps them in osmotic adjustments, maintains protein structures, and stabilizes the cellular membranes20. Also, these organic osmolytes protect the plants from the chaotropic impacts of cations by regulating the cell water status21.

Modifications in leaf internal architecture are considered a most imperative characteristic of salt-tolerant plants to circumvent salt-induced oxidative damage22. Halophytes change the palisade mesophyll structure by forming large chloroplasts, which directly confers efficient photosynthesis23. Salt-tolerant ecotypes own increased leaf and stem succulence by an increase in the midrib and lamina thickness that enhances water storage capacity and is the most important criterion to evaluate the cell water status24,25. During salt-induced osmotic stress, plants possess a thick epidermis, which acts as a physical barrier to reduce the water loss from the leaf surface26,27. Also, an increase in these ectodermal tissues plays a crucial role under higher salt intensities in halophytes and causes alteration in ionic pathways28. The alterations in the leaf epidermis greatly contribute to salt localization and increase in the xylem area for uniform distribution of Na+ in leaves for ion homeostasis29. Halophytes are equipped with epidermal components trichomes, mainly unicellular or multicellulars, to eliminate excessive salts, protect from herbivores, and restrict the entry of pathogens30. Under extremely arid conditions, halophytes develop the trichomes/ epidermal bladder cells (EBCs) for the Na+ sequestration due to small vacuoles31,32. The presence of dermal microhairs (trichomes) on adaxial/abaxial leaf surfaces seems to sequester the excess salts away from the leaf’s metabolically active epidermal and mesophyll cells33.

Fimbristylis complanata is a halophytic sedge inhabiting salt-affected lands, saline wetlands, and salt marshes. This halophytic species has been explored less, and rare literature regarding their salt tolerance and adaptive mechanisms for successful growth in saline areas is available. To our knowledge, the modification in growth, photosynthesis, and leaf anatomy at various salt intensity and incubation phases has not been investigated earlier in Fimbristylis plants. In our previous work, we mainly focused on the intensity of salt and directly subjected salt stress to Fimbristylis complanata without consideration of the time. This is the first detailed study in which we have checked the salt tolerance potential of Fimbristylis complanata under temporal changes. This species is selected because of its wide distribution on salt-affected aquatic lands and saline marshes. Also, often, this specie is regarded as a potential candidate for the rehabilitation/ phytoremediation of the salt effect marginal wetlands. These observations provoked our interest in designing the present study to evaluate the rapid modification in growth, photosynthesis, and internal leaf architecture with salt intensities and short incubation phases (30 days). This study provided useful insight into the current study and deciphered future interest in deeper studies to explain the current modifications in growth, which are probably involved in ROS and nitric oxide production and changes in hormone homeostasis.

Materials and methods

Collection sites

Two ecotypes of aquatic Fimbristylis complanata were collected from salt-affected wetlands of Punjab, Pakistan, viz. SH-Sahianwala (ECe 46.98 dS m−1) a hypersaline salt marsh (31° 36′ 08.45″ N, 73° 14′ 56.98″ E, altitude 191 m a.s.l.) having a clayey soil; HR- Rasool Headworks (ECe 26.01 dS m−1) seasonal inundations along the riverside with mixed sand and clayey type of soil. The soil physicochemical characteristics of these collection sites mentioned in our earlier work29.

Plant culture, growth conditions, establishment, and layout

Ecotypes of Fimbristylis complanata were established in plastic pots (Diameter 30 cm, height 45 cm for each) in the Research area of the old botanical garden (31° 25′ 44″ N, 73° 4′ 18″ E, altitude 186 m a.s.l.) to acclimatized (for 30d) with the natural atmosphere of Faisalabad. Six plantlets (rhizomes of 6 cm in length at the earlier growing stage) were separated from the pots and grown in individual pots half-filled with a mixture of sand and clayey loam soil with a ratio of 1:1 having pH 6.7 and irrigated with 2L of water. The present experiment was laid out under natural environmental conditions within three replicates and three salt regimes. The average day and night range 36–40 °C and 25–30 °C, respectively, and the photoperiod ranges 13–14 h with relative humidity (RH) ranging from 56.7 to 57.9% on average.

Treatment applications

After 30 days of plant establishment, salt treatments were applied under varying levels (0, 200, 400 mM NaCl) with three time intervals (i.e., 0, 15, and 30 days). Here, 0-day means the 30th day of establishing ecotypes in a natural environment of experimentation before the imposition of salt stress. The 0 mM was designated as an absolute control (ECe 0.012 dS m−1). In comparison, NaCl treatments were progressively increased by adding 50 mM NaCl day−1 increments to prevent sudden osmotic shock and maintain the desired concentrations. After 0, 15, and 30 days (d) of stress implications, plants were carefully uprooted and thoroughly washed with tap water to assess the growth, photosynthesis pigments, and leaf anatomical features.

Plant analysis and measurements

Measurement of growth traits

To determine the shoot fresh weight (SFW) and shoot dry weight (SDW), three plants from each pot were uprooted carefully on days 0, 15, and 30 days from each treatment and washed with tap water to remove the soil and sand particles. Freshly harvested plant samples were weighed to determine SFW, and these plants were placed in an oven at 80 °C for 48 h until a constant weight was achieved to assess the SDW. Leaf area was recorded using the formula ((length × width × correction factor 0.75). Root length and plant height were recorded using a cm scale. The relative growth was measured using RGR = LAR × ULR, here ULR-unit leaf area and LAR- leaf area ratio. The leaf area ratio (LAR) was calculated using the ratio between the plant’s total leaf area and SDW using the equation LAR = LA/SDW. The leaf mass fraction was calculated by LDW/SDW; LDW means leaf dry weight and SDW shoot dry weight of the plants. The mean leaf area displayed per unit of leaf weight was measured by SLA = LA/LW; here SLA means specific leaf area, LA is the total leaf area and LW means the weight of the leaf. Unit leaf rate was determined by the formula ULR = 1/LA/ SDW/ dt; here, LA is leaf area, SDW was shoot dry weight, and dt was the increase of leaf dry weight within unit per area in a unit time.

Leaf photosynthetic pigments

Leaf photosynthetic pigments, such as chlorophylls a and b, were estimated using the Arnon method34, and carotenoids using the Davis method35. Fresh leaf samples (500 mg) were taken after 0, 15, and 30 days from each salt treatment, pulverized at 30% V/V at 4 °C, and left overnight in dark conditions. Homogenized material was centrifuged at 10,000 X g for 5 min, and absorbance was recorded at 663, 645, and 480 nm with the help of UV- a visible spectrophotometer (Hitachi, 220 Japan). An appropriate formula ascertained the pigment contents.

Photosynthetic traits

Photosynthetic traits were determined using the first fully mature expanded leaf from each treatment plant at 0, 15, and 30 days using an infrared gas analyzer IRGA (LCA-4, ADC, and Hoddesdon, England). Measurements of net CO2 assimilation rate, stomatal conductance, transpiration rate, and water use efficiency were recorded under moderate light availability between 7:30 to 8:30 h. The portable photosynthetic system has a leaf chamber (2 × 3 cm2), CO2: 400 ppm, relative humidity (RH): 50–60%, rate of flow: 300 µmol s−1, photosynthetically active radiations (PAR): 700 µmol m−2 s−1 and vapour pressure were less than 2. To obtain the constant reading, leaves were placed for a sufficient time in the chamber.

Anatomical traits

A fully expanded leaf was separated from the plant body to study the anatomical traits. The leaf material was fixed in a formalin acetic acid solution containing acetic acid 5%, formalin 10%, ethyl alcohol 50%, and distilled water 35% v/v as a fixative. After 48 h, plant material was transferred into acetic acid and alcohol solution (ethanol 70% and 30% acetic acid) for prolonged storage. Permanent slides were prepared by freehand sectioning and series dehydration using the ethyl alcohol to study the microstructure, and the double staining method was followed (stains were fast green and Safranine). Microphotographs were taken on a camera-equipped microscope (Nikon, 104, Japan). Leaf blade microstructure such as leaf midrib, lamia, bulliform cell, epidermal cell, cortical cell thickness, epidermal cell, vascular bundle, metaxylem, phloem, and aerenchyma area were measured by a calibrated ocular micrometer.

Statistical analysis

The present experiment was designed as a completely randomized design (CRD) with three replications. Statistical analysis and data visualization were performed using R statistical software36 through the R integrated development environment RStudio36. The data were subjected to a two-way analysis of variance (ANOVA) at a significance level (p ≤ 0.05) to test the effect of NaCl treatment on each ecotype on each ecotype within different periods and the interaction between SH and HR ecotypes. Treatment means and standard errors (SE) for each ecotype, salt treatment, and period were compared using Tukey’s HSD (honestly significant difference) test, utilizing the ‘agricolae’ package in R software37. Additional analysis was comprised of eclipsed principal component analysis using “FactoMineR and ggplot2”, as well as clustered heatmaps and scatter correlation matrices using “pheatmap” and “GGally” codes. A cutline-grouped dendrogram was constructed using the customized Ward.D2 method for hierarchical clustering and cuts in R software37. It employs R libraries ggdendro, factoextra, ggplot2 and applies the cut method with significance levels based on simulated p-values.

Results

Growth attributes

Shoot fresh weight (SFW) and shoot dry weight (SDW) of ecotype SH were significantly decreased from 0–30 days incubation at moderate salinity (200 mM NaCl). However, this ecotype increased SFW and SDW at 400 mM NaCl. In ecotype HR, on day 15th, the SFW was significantly increased in response to high salt concentration (400 mM NaCl), while an obvious reduction occurred in SDW in this case (Table 1). The leaf area (LA) of the SH ecotype decreased at moderate salt stress from 0–30 days, while it significantly increased at a high salt level (400 mM) with an increase in duration. The ecotype HR showed a significant increase in LA with increased salt intensity and duration. This increase was more pronounced at 400 mM NaCl on the 30th of the day (Table 1). Salt stress showed no change in root length (RL) in response to long-term exposure and high salt concentration (400 mM) in the SH ecotype. However, the HR ecotype exhibited an increase in RL at 400 mM NaCl on the 30th day (Table 1). Salt levels significantly increased plant height (PH) in SH and HR ecotypes. Long-term exposure to 400 mM NaCl significantly increased the PH in both ecotypes of F. complanata. The relative growth rate was significantly reduced due to long-term salt exposure in both ecotypes of F. complanata (Table 1). The long-term exposure to 400 mM NaCl increased the leaf mass fraction (LMF) in the HR ecotype, while a reduction occurred in the specific leaf area of the SH ecotype. Increasing salt levels reduced the unit leaf area from early to long-term exposure (Table 1).

Table 1 Growth traits of F. complanata grown under control (0 mM NaCl), moderately saline (200 mM NaCl), and highly saline (400 NaCl) conditions for variable time durations.
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Photosynthetic pigments

Chlorophyll a (Chl a) increased at moderate salinity from (15–30 days), while reduced at high salinity (400 mM) in the HR ecotype. In the SH ecotype, the Chl increased in response to long-term exposure and increasing levels of NaCl (Fig. 1a). The concentration of Chl b in the HR ecotype was highest at a salt concentration of 400 mM NaCl on day 0, but it showed a decreasing trend from day 15 to day 30. The mean values for Chl b were lower at day 0; however, a substantial increase occurred at 400 mM NaCl concentration from day 15-30th in the SH ecotype (Fig. 1b). In the HR ecotype, a substantial carotenoid reduction was observed from days 15–30 in response to increasing salt regimes. The SH ecotype showed a higher amount of carotenoids at 400 mM NaCl level on day 15th; however, no significant change occurred on day 30th with increasing salt levels (Fig. 1c).

Fig. 1
figure 1

Photosynthetic pigments of Fimbristylis complanata ecotypes [Rasool Head Works (HR), Sahianwala (SH)] treated with different salt levels for different periods. Means ± SE are provided. Bars sharing the same lowercase letters are not significant (p ≤ 0.05) for different salinity treatments applied to an ecotype at a specific exposure time.

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Photosynthetic traits

Net CO2 assimilation rate in HR ecotype slightly increased at moderate salt-stressed (200 mM) on day 15th, while the high salt level on day 15th exhibited no change, and a drop occurred on the 30th day in response to 400 mM NaCl level (Fig. 2a). The HR ecotype transpiration rate (A) increased at a higher salt intensity on the 15th day, declining on the 30th. In the SH ecotype, E increased with an increase in salt intensity on the 15th day, whereas it decreased on the 30th day (Fig. 2b). The stomatal conductance (gS) exhibited a decreasing trend in HR ecotype, and obvious reductions were observed on day 30th at higher saline regimes of 400 mM NaCl. The SH ecotype also showed a maximum decrease in gS at higher salinity (400 mM NaCl) on the 30th day of salt exposure (Fig. 2c). The water use efficiency (A/E) increased from the 0–15th day with increasing NaCl intensity while remaining unchanged at 400 mM NaCl on the 30th day in the HR ecotype. The A/E increased in SH ecotype on the 15th day while exhibiting no change on the 30th day in response to high salt concentration (Fig. 2d).

Fig. 2
figure 2

Photosynthetic and gas exchange attributes of Fimbristylis complanata ecotypes [Rasool Head works (HR), Sahianwala (SH)] treated with different salt levels for different periods. Means ± SE are provided. Bars sharing the same lowercase letters are not significant (p ≤ 0.05) for different salinity treatments applied to an ecotype at a specific exposure time. For abbreviation, see the description list at the start of the manuscript.

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Leaf anatomical architecture

Leaf midrib thickness (MrT) responded to the maximum increase in MrT that occurred on the 30th of the day and at a high NaCl concentration of 200 mM. However, a reduction in MrT has occurred in response to high salt concentration. The HR ecotype showed an increase in MrT in earlier days (0–15 days) while reduced significantly on day 30th in response to 400 mM NaCl. In earlier days (0–15), the leaf thickness (LTh) decreased with increased salt levels; however, it showed a significant increase on day 30th at a high salt level of 400 mM in both ecotypes. The bulliform thickness (BTh) increased significantly in the SH ecotype at 200–400 mM NaCl from days 15–30th as compared to the 0th day. The BTh in the HR ecotype exhibited a significant increase from 0–30th day at 200 mM salt concentration, while a substantial reduction was noticed at 400 mM NaCl. However, the BTh increased with NaCl concentration, and the maximum increase was found on day 30th. The ECA increased with the increase in salt levels and duration. The ECA was the maximum in SH at 400 mM NaCl on the 15th day. The MXA of leaves was decreased in response to moderate salt intensity, while a significant increase occurred on day 30th in exposure with increasing salt regimes. The MXA in the SH ecotype showed a reduction in response to higher salt concentrations and maximum duration. The PhA responded negatively as it reduced in both ecotypes from days 0–30th. On day 30th, the SH ecotype secured an increase in PhA with an increase in NaCl levels. The CcT and ArA of the HR ecotype increased in normal and moderate stresses from day 0–30, while SH did not respond to a significant change in both traits (Table 2; Fig. 3,4).

Table 2 Leaf anatomical traits of F. complanata grown under control (0 mM NaCl), moderately saline (200 mM NaCl), and highly saline (400 NaCl) conditions for variable time durations.
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Fig. 3
figure 3

Leaf anatomical modifications in Rasool Headworks (HR) population of Fimbristylis complanata treated with different salt levels for different time intervals. a. (control): Triangular leaves with large extensively sclerified chlorenchyma (Ch) and vascular bundles (VB), enlarged lysigenous aerenchymatous (Ae) cavities and cortical parenchymatous (P) region; b. (moderately saline): Extremely large vascular bundles (VB) with enlarged phloem area (Ph), aerenchymatous (Ae) cavities, and cortical parenchyma (P); c. (highly saline): Greatly reduced midrib (Mr), vascular (VB), cortical parenchymatous (P) regions and aerenchymatous area (Ae); d.(Control): Thick midrib with large chlorenchyma (Ch) and cortical parenchyma (P), sclerified vascular bundles (VB) and aerenchymatous (Ae) cavities with reduced size; e. (moderately saline): Sclerified and enhanced midrib thickness and vascular bundles (VB), larger cortical parenchyma (P), lower epidermal thickness (LE) and aerenchymatous (Ae) cavities; f. (highly saline): Significantly thicker lower epidermis (LE), reduced vascular bundles (VB) and cortical parenchyma (P), enlarged metaxylem vessels (Mt) and aerenchymatous cavities (Ae); g. (control): Thick leaves with an increased size of vascular bundles (VB) and aerenchymatous (Ae) cavities; h. (moderately saline): Decreased midrib thickness, vascular bundle (VB) size and cortical parenchyma (P), lamina size (L) significantly increased; i. (highly saline): Maximum leaf thickness with larger lamina (L); high midrib thickness (Mr), intensively sclerified vascular bundles (VB), large aerenchymatous cavities (Ae) and lower epidermal (LE) thickness.

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Fig. 4
figure 4

Leaf anatomical modifications in Sahianwala (SH) population of Fimbristylis complanata treated with different salt levels for different time intervals. Descriptions: Sahianwala (SH): Day-0. a (control): Thick leaves with increased upper epidermal (UE) thickness and aerenchymatous cavities (Ae), reduced vascular bundle (VB); b (moderately saline): Greatly enhanced leaf thickness having large vascular bundles (VB), aerenchymatous cavities (Ae) and thicker upper epidermis (UE); c (highly saline): Thickest leaves with large vascular bundles (VB), aerenchymatous cavities (Ae) and thicker upper epidermis (UE), metaxylem vessels (Mt) extremely large. Day-15. d (control): Small and narrow leaves with large vascular bundles (VB), reduced upper epidermal (UE) thickness and large aerenchymatous cavities (Ae); e (moderately saline): Increased leaf thickness and vascular bundles (VB), metaxylem area (Mt) and upper epidermal (UE), very reduced aerenchymatous cavities (Ae); f (highly saline): Extremely thicker leaves consisting of smaller vascular bundles (VB), thicker upper (UE) and lower epidermis (LE), Day-0. g (control): Thickest leaves comprising large vascular bundles (VB) and aerenchymatous cavities (Ae); enhanced upper epidermal (UE) thickness, reduced lower epidermal (LE) thickness; h (moderately saline): Thicker leaves with more developed parenchyma (P) below the upper epidermis (UE), vascular bundles (VB) large with widened metaxylem vessels (Mt); i (highly saline): reduced leaf thickness, vascular bundles area (VB), metaxylem area (Mt), development of additional parenchyma (P) layer below thick upper epidermis (UE).

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Epidermal surface anatomical traits

The AbSA has significantly increased in the SH ecotype with long-time exposure to salt stress due to increased NaCl treatment levels. The AbSA in the HR population showed a reduction with an increase in salinity and duration. The AdSA in SH and HR ecotypes was significantly reduced by the rise in salt duration and intensity of NaCl. The AbSD and AdSD increased in SH and decreased in HR during the salt incubation periods at moderate to higher salt concentrations. However, no significant change occurred in AdSD at higher salt concentrations in the HR ecotype from days 0 to 30. (Table 3; Fig. 5,6).

Table 3 Leaf epidermal traits of F. complanata grown under control (0 mM NaCl), moderately saline (200 mM NaCl), and highly saline (400 NaCl) conditions for variable time durations.
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Fig. 5
figure 5

Lower epidermal modifications showing stomatal features (S) concerning salt tolerance of Fimbristylis complanata ecotypes treated with different salt levels for different periods. Descriptions: Rasool Headworks (RH): Day-01. a (control): Large stomata arranged in two lines between minor vein; b. (moderately saline): Small and circular stomata arranged in more than two lines; c. (highly saline): Small and narrowly elliptical stomata arranged in two lines between minor veins. Day-15. d (control): Large and elliptical stomata arranged irregularly on the whole epidermis; e (moderately saline): Numerous rhomboidal stomata having irregular pattern; f (highly saline): More or less circular stomata covering most of the epidermal surface area. Day-30. g (control): Few and smaller stomata arranged in single file between minor vein; h (moderately saline): More or less elliptical stomata arranged in irregular pattern; i (highly saline): large Rhomboidal stomata covering most of the epidermal surface. Sahianwala (SH): Day-0. j (control): Small and numerous stomata with more or less circular shape; k (moderately saline): Few and large stomata having more or less elliptical shape, arranged in three files; l (highly saline): Round stomata arranged in irregular pattern. Day-15. m (control): Few elongated stomata irregularly arranged on an epidermal surface; n (moderately saline): Few large elliptical stomata arranged in two files between minor vein; o (highly saline): Few elongated stomata arranged in two files between minor vein. Day-0. p (control: Few large stomata regularly arranged in two files between minor vein; q (moderately saline): Few rhomboidal and large stomata, arranged in irregular pattern; r (highly saline): Small, numerous stomata and more or less rounded stomata arranged in irregular pattern between minor vein.

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Fig. 6
figure 6

Upper epidermal modifications showing stomatal (S) and other features concerning salt tolerance of Fimbristylis complanata ecotypes treated with different salt levels for different periods. Descriptions: Rasool Headworks (RH): Day-01. a (control): Few, small and elliptical stomata arranged in two or more lines; b. (moderately saline): Small and rounded stomata arranged in one line between minor vein; c. (highly saline): Few, small, and more or less rounded stomata arranged irregularly. Day-15. d (control): Few and small stomata with more less elliptical shape; e (moderately saline): Small and pounded stomata arranged in irregular pattern; f (highly saline): Rudimentary small stomata arranged in two or more lines between minor vein. Day-30. g (control): Few and large stomata with a less circular shape, arranged irregularly between minor veins; h (moderately saline): Very few and small stomata arranged roughly between minor vein; i (highly saline): Small and rounded stomata arranged in two lines irregularly between minor vein. Sahianwala (SH): Day-0. j (control): Large, more or less elliptical stomata arranged irregularly between minor vein; k (moderately saline): Few and large stomata arranged coarsely between the minor vein; l (highly saline): Very few and elongated stomata arranged in an irregular pattern. Day-15. m (control): Large and circular stomata deeply seated between the minor vein; n (moderately saline): Less number of elongated stomata, arranged in a single line between the minor vein; o (highly saline): Large and more or less elliptical stomata arranged in two or more lines. Day-0. p (control: Few rhomboidal stomata arranged very coarsely between minor vein; q (moderately saline): Large, more or less circular stomata arranged irregularly between minor vein; r (highly saline): Large, more or less elliptical stomata arranged in two lines between minor vein.

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Multivariate analysis

Principal component analysis (PCA)

The principal component analysis illustrated the effect of salt intensity and duration on the growth and photosynthetic pigments of SH and HR ecotypes. A closer distance between eigenvectors in the same direction indicated a significant association of the variable components in discriminating salt incubation and intensity. The PCA biplot for these traits was more influential, with a cumulative contribution of 54.3% variation. Variables LW, SFW, Chl a, and LA were strongly associated and clustered toward the positive side of PCA1 and positive eigenvalues. The LA was closely associated with moderate saline exposure (D2). The LW was grouped with D3, SFW, and Chl a was linked with the D1 (Day-0). The growth traits LAR and LMF excelled towards the PCA2 with a strong association and higher positive eigenvalues under D3. The RGR and ULR were lowered in response to more salinity duration by showing the negative eigenvalues (Fig. 7a, Table 4).

Fig. 7
figure 7

PCA biplot of (a) growth, photosynthetic pigments, and gas exchange attributes, (b) leaf anatomical traits and leaf epidermal traits of Fimbristylis complanata ecotypes treated with different salt levels for different periods (D1; Day-0, D2; Day-15, D3; Day-30). For abbreviation, see the description list at the start of the manuscript.

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Table 4 Component weights (PCAs) for growth, photosynthetic pigments and gas exchange traits of Fimbristylis complanata.
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The explained variations of PCA for leaf anatomical and epidermal surface were 25.3% and 39.6% (Total 64.9%) between time and intensity. The PhA, ArA, and CcT excelled in a strong association with higher positive eigenvalues, which increased significantly and eclipsed on Day 15 (D2) and Day 30 (D3). All of these traits were more concentrated on the positive side of PCA1. These traits are also closely loaded, along with moderate exposure to salt, as in Day 15 (D2). The variables BTh, ECA, ETh, and MXA showed a negative loading of eigenvalues toward the PCA1 with a reduction pattern. These traits were strongly associated with moderate to high salt exposure (D2-D3). The AbSD was strongly associated with moderate salt exposure (D2) with a higher positive eigenvalue (+ 4). The LTh and AdSA were loaded toward the PCA2 along a higher positive eigenvalue. The AbSA and AdSD were plotted in the center and showed no significant influence and contributed toward maximum salt exposure (Fig. 7b, Table 5).

Table 5 Components weights (PCAs) for leaf anatomical and epidermal traits of Fimbristylis complanata.
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Clustered heatmaps

A heatmap was constructed to represent the influence of salt intensity and duration on the growth, photosynthesis, and photosynthetic pigments of both ecotypes (Fig. 8a). Growth traits such as SDW and PH were significantly (p ≤ 0.05) associated with HR ecotype (E2) under moderate salt exposure (D2) at 200 mM NaCl (T2). However, both growth traits in SH ecotype (E1) negatively contributed to day 30th and 400 mM concentration and showed a reduction at higher salt exposure and duration. The SFW and LW of both ecotypes (SH-E1; HR-E2) were strongly associated at D1, D2, and D3 under 200 mM (T1). The ecotype E1 (SH) secured higher SFW and LW in response to greater salt intensity (T2) at maximum salt exposure (D3). The ULR was strongly linked with D1 (Day-0) and D2 (Day-30) in absolute control (T0). A slight increase occurred in E1 (SH) at higher salt exposure (T2). The photosynthetic traits WUE and Pn showed a strong influential pattern in response to 400 mM NaCl (T2) at maximum salt exposure D3 (Day-30) time. A strong grouping was shown between PH: SDW, E: Chl a SLA: Caro, and LAR: LA. In general, all photosynthetic traits positively influenced the growth traits of F. complanata by showing a clustering.

Fig. 8
figure 8

Clustered heatmaps representing the influence of salt intensity and duration on a) growth, photosynthetic pigments, and gas exchange attributes and b) leaf anatomical and leaf epidermal traits of Fimbristylis complanata ecotypes. E1-Sahianwala, E2-Rasool Headworks, D1; Day-0, D2; Day-15, D3; Day-30, T0-0 mM NaCl, T1-200 mM NaCl, T2-400 mM NaCl. For abbreviation, see the description list at the start of the manuscript.

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A clustered heatmap represented a strong association of salt exposure and intensity on leaf anatomical traits of F. complanata (Fig. 8b). The MXA of E1 (SH) was strongly associated with the D1 and T1 (200 mM) salt concentrations. The PhA of E1 (SH) strongly corresponded to D2 (Day-15) under moderate salt regimes (T2-200 mM). The leaf anatomical traits such as CcT and ArA of E1 (SH) responded better under moderate salinity exposure (D2) and intensity (T2). The AbSD of E1 (SH) strongly contributed to D2 (Day 15) and T1 (200 mM). The AbSA and AdSD of SH (E1) were positively influenced by D1-D3 under both salt levels (T0, T1). The VBA of E2 (HR) was negatively linked with the moderate (T1) and higher salt regimes (T2). A strong clustering was indicated between AbSA: AdSD, ETh: BTh, and AdSA: AbSD.

Pearson correlation matrix and scatter plot

The Pearson correlation matrix drew a significant (p ≤ 0.05) positive correlation for growth, photosynthesis, and photosynthetic pigments of F. complanata under variable salt intensity and duration (Fig. 9). A strong positive correlation was assessed between Chl a, b with SFW (r = 0.63) and SDW (r = 0.72), LW (r = 1.00) with RL (r = 0.60) and Pn with Chl b (r = 0.53). A strong negative correlation was observed between LMF (r = −0.75) and PH (r = −0.66), as well as between LAR and PH (r = -0.66), ULA (r = -0.40), and LW (r = 0.41). The ULR was negatively skewed in the scatter plot with growth traits such as SFW, SDW, PH, RL, LW, and SLA. The fitted line between ULR and RGR was more skewed with a positive curve. The central diagonal pattern of ULR, along with all other traits, was steep. The leaf anatomical traits had significant (p ≤ 0.05) association and excelled through the Pearson correlation matrix (Fig. 10). The ECA was strongly correlated with CcT (r = 0.70) and ArA (r = 0.62). These traits had a lesser skewed fitted line in the scatter plot. The ArA (r = 0.42) was strongly and positively correlated with CcT (r = 0.80) with a most skewed fitted line of a scatter plot. The AbSA (r = 0.88) and AdSA (r = 0.46) were strongly associated in the correlation matrix. The strong negative correlation was assessed between VBA (r = −0.27): LTh (r = −0.37), PhA (r = −0.43): CcT (r = −0.47). In the scatter plot, the LTh and BTh had a linear fitted line with larger intercept values. The CcT and ArA showed a more skewed curve with a higher positive correlation.

Fig. 9
figure 9

Pearson correlation matrix and scatter plot of growth, photosynthetic pigments, and gas exchange traits of Fimbristylis complanata ecotypes treated with different salt levels for different periods (D1; Day-0, D2; Day-15, D3; Day-30). In each figure, the top right panel of the plot shows the correlation coefficient (r) and level of significance (p ≤ 0.05), and the bottom left of the plot shows the bivariate plot with fitness of the regression line. The distributions of various traits are shown in the central diagonal line and traits on the marginal axis. Red represents a higher degree of positive correlation, while blue represents negative correlation. For abbreviation, see the description list at the start of the manuscript.

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Fig. 10
figure 10

Pearson correlation matrix and scatter plot of leaf anatomical and leaf epidermal traits of Fimbristylis complanata ecotypes treated with different salt levels for different periods (D1; Day-0, D2; Day-15, D3; Day-30). In each figure, the top right panel of the plot shows the correlation coefficient (r) and level of significance (p ≤ 0.05), and the bottom left of the plot shows the bivariate plot with fitness of the regression line. The distributions of various traits are shown in the central diagonal line and traits on the marginal axis. Red represents a higher degree of positive correlation, while blue represents negative correlation. For abbreviation, see the description list at the start of the manuscript.

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Hierarchical clustered dendrogram

The hierarchical clustering dendrogram exemplifies the clustering of both ecotypes E1-Sahianwala E2-Rasool headworks under different salt regimes and periods. This clustered map provides insight into how ecotypes responded to salt stress. The first clustered dendrogram demonstrated a strong linkage of varying NaCl concentrations (T0, T1, and T2) over different periods (D1, D2, D3) with various growth and physiological attributes. Also, ecotypes from Sahianwala (E1) and Rasool headworks (E2) exhibited a divergent response to salt concentrations and exposure time. The variable E1D1T1 was strongly diverged with E2D1T1, E2D1T0, E1D3T0 and E2D3T0. This E1D1T1 also shows a distinct linkage to cutline groups plotted on the right side of the hierarchical plot. Moreover, this plot may illustrate the divergent or similar responses, which depend on the stress level and time. A clear-cut differentiation was assessed between T0, T1, and T2 salt-treated groups, reflecting the progressive responses to E1 and E2 ecotypes.

The second dendrogram showed how the leaf’s anatomical and epidermal traits influenced the varied salt regimes under temporal conditions. This plot indicates the anatomical traits strongly link saline levels and duration. The observable changes progressed from day 0 to day 30 (D1-D3); however, separating E1 and E2 ecotypes highlights differential tolerance. The E1D1T1 was tightly linked with E1D1T1, E1D1T2, and E1D2T1 while showing a sub-grouping with all other salt concentrations and durations. Both plots effectively captured the complex nitration among physio-biochemical traits and leaf microstructures in both ecotypes.

Discussion

The growth pattern of the plants is an important tool to screen out salt-tolerant ecotypes during salt exposure38,39. In the present work, maximum plant biomass (SFW, SDW) was observed in the SH ecotype at 400 mM NaCl concentration on the 30th day. The increase in biomass in salt-tolerant ecotypes occurs due to the accumulation of osmoprotectants and photosynthetic pigments. It allows salts to act as osmotic agents to maintain the turgor and growth, ultimately leading to increased biomass. Many halophytes can easily survive in high-saline environments by improving biomass productivity40. Higher plant biomass in halophytic sedges indicates a potential for higher salt tolerance18. The leaf area (LA) of the SH ecotype decreased from days 0-30th in response to 0–200 mM salt concentrations, while a slight increase occurred at 400 mM NaCl. The ecotype HR showed an increase in LA at 400 mM NaCl on day 30th. An increase in the leaf area of SH ecotypes acts as an adaptive component for salt tolerance to optimize photosynthesis, regulate photosynthesis and WUE, and dilute internal salt concentrations. A significant increase in LA is directly linked with the net photosynthesis of the whole plant41.

Halophytes increase LA to enhance photosynthesis and tolerate the salt extremities18,42. The efficient photosynthesis in halophytic plants directly anticipates better growth31. Acclimation of root growth in halophytes is considered a strategic dynamic to regulate growth and is linked with the expansion of the leaf43. On day 30th, the RL in the HR ecotype increased at 400 mM NaCl. The plasticity in root growth supports plants in saline conditions44. The high salt intensity and long-term exposure to salinity caused an increase in plant height (PH) in both ecotypes under moderate to high salts. Halophytic plants exhibited increased plant height to increase the biomass when exposed to moderate salt exposures45. In the SH ecotype, the PH increased with increased salt regimes but reduced with time. In the SH ecotype, the initial increase in plant height with rising salt regimes followed by a reduction over time may be due to the cumulative stress of prolonged high salinity, which eventually overwhelms the plant’s adaptive mechanisms to conserve the resources46. The PH showed an increasing trend with increased salt levels and duration in the HR ecotype. This increase indicates enhanced salt tolerance and efficient adaptive mechanisms due to large epidermal tissues in the leaf sequestering salts to sustain under prolonged salinity. Also, salt-tolerant plants compartmentalize Na+ in vacuoles to synthesize organic osmolytes for osmotic adjustments. The relative growth rate (RGR) was reduced in both ecotypes of F. complanata at long-term exposure to 400 mM NaCl. Prolonged exposure to salinity reduced the photosynthetic activity of plants due to stomatal limitations and photochemical damage in mesophyll cells47. The reduction in RGR also reflects the adaptive mechanisms to successfully thrive in saline extremes by conserving energy and resources. Ecotypes also prioritize osmotic balance over rapid growth and salt exclusions by shoots and roots. This adaptation enhances their survival in saline conditions despite reducing overall growth and height. The leaf mass fraction (LMF), specific leaf area (SLA), and unit leaf area (ULA) decreased in response to prolonged exposure to high salinity (400 mM NaCl). A reduction in RGR occurred due to a decline in net CO2 assimilation37 and stomatal conductance on day 30th. This reduction might be attributed to the reduction of LMF, SLA, and ULA of F. complanata. Such reduction in these growth traits was previously reported in many salt-tolerant types of grass, such as Panicum virgatum and Arundo donax48, and in Phragmites australis49.

The results of photosynthetic pigments suggest the SH ecotype is more salt tolerant than its counterpart, the HR ecotype. In less tolerant halophytes, high salinity destroys photosynthetic pigments, while in salt-tolerant plants, these pigments increase for enhanced salt tolerance50. Outwardly, the salt-tolerant ecotypes showed an increase or no change in photosynthetic pigments (Chl a & b), which reduced salt-sensitive ecotypes. The increase in carotenoid contents might be a function of excessive energy dissipation under salt stress51.

The net CO2 assimilation of halophytic F. complanata SH ecotype enhanced under 200 mM concentration with durations. The Pn in halophytes either remains unchanged or enhanced37,52. Sustaining the Pn under high salt increments relates to efficient storage of salts in vacuoles and dilution effects through increased leaf succulence, which protects the chlorophylls53. Pn of HR ecotype reduced at prolonged duration and high salt regimes. Salt stress induces the closure of stomata, causes a reduction in Pn, and impairs the photosynthetic apparatus of plants54. The initial increase in Pn of SH ecotype to moderate salinity exposure is concerning improvement in the WUE of this ecotype to sustain the photosynthetic performance, while the reduction in HR ecotype at higher saline increments coupled with lower gs, as previously observed in Kochia prostrata L. to higher salt levels55. The transpiration rate (E) in both ecotypes of F. complanata decreased at 400 mM after day 30th of salt exposure. The dose-dependent reduction in E in salt-tolerant species to maintain cell water status and enhanced uptake of CO2. This reduction occurs due to stomatal limitations in reducing energy usage and maintaining cell internal osmotica and local ABA synthesis55,56. Halophytes may employ a decrease in E to avoid excessive dehydration, prevent salt translocation to leaves, and enhance the photosynthetic machinery of plants57. Stomatal conductance (gs) in both ecotypes decreases with increased salt levels and duration. In halophytes, the stomatal conductance reduces in a dose-dependent manner56, as revealed in the present study. The stomatal conductance and CO2 assimilation exhibited a sigmoid reduction pattern in response to high salt regimes. Also, reduction in the gs is an immediate response of the halophytes to elevated levels and prolonged exposure to salts by modulations of stress signaling pathways to enhance the stomatal closure, minimize the water loss, and limit the uptake of the harmful ions to maintain cellular homeostasis58. In halophytes, gs are directly coupled with Pn, which implies that plants retain sufficient water without penalty for biomass and photosynthesis59. The water use efficiency (A/E) of the SH ecotype increased at high salt exposure. Halophytes sequester Na+ in vacuoles and control stomatal numbers to optimize A/E efficiently in stressful environments60.

Leaf midrib thickness (MrT) and leaf thickness (LTh) in both ecotypes increased on day 15th in response to 400 mM salt level. An increase in MrT and LTh mediates leaf succulence to enhance the flow of materials in conducting tissues and water storage capacity 61,62,63. In both ecotypes, leaf bulliform thickness (BTh) increased from earlier to a prolonged period. Enlarged and well-developed bulliform cells in leaves efficiently roll the leaf to prevent the plant from dehydration24,64. Leaf rolling under salt stress is considered more convenient for better photosynthesis65. An increase in the epidermal cell area in the SH ecotype and an increase in ECA protect the plant from the outer environment and alter the ionic pathways in halophytic species28. It also consists of large vacuoles for the compartmentalization of salts63. Epidermal cells in halophytes undergo two to three rounds of division to form an epidermal extension to counteract the salt-induced osmotic stress involved in the salt exclusions, which greatly contribute to survival in salt extremes33. Glandular trichomes are bladder-like hairy projections that reduce transpiration and involve salt secrtions66. The metaxylem cell area (MXA) increased in HR and remained unchanged in the SH ecotype. This increase in MXA contributes to better conduction of salts and water under osmotic stresses and increases the probability of embolism under severe salt67. The MXA indicates a salt tolerance in halophytic sedges by maintaining ion homeostasis28. The phloem area (PhA) responded negatively with a decline in response to prolonged exposure, while on earlier days, it increased at high salt regimes. Enlargement in PhA facilitates photosynthetic products and nutrients across the plant body68. The cortical cell thickness (CcT) and aerenchyma area (ArA) responded negatively to time duration while showing increased salt levels in both ecotypes. The increase in the cortical parenchyma of the leaves contributes to a broader leaf sheath, an important commodity for water conservation69,70. Large aerenchyma stores surplus water and gases and enhances salt tolerance29, protecting photosynthetically active cells71 and fast diffusion of solutes72.

The abaxial stomatal area (AbSA) increases in SH ecotype while reducing the HR ecotype of F. complanata under higher salinities. Stomata with a small surface area responded more frequently to salt stress. Salinity-induced osmotic stress and reduction in the stomatal area are beneficial for the plants because they reduce the expenditure of energy73. Reduction in the stomatal area is also helpful in reducing the transpiration rate during stress74. The AdSA of both ecotypes increased along the higher salinities and duration75. The abaxial and adaxial stomatal increased at moderate salinity and duration, while there was no change at higher and prolonged salinities. Smaller stomata are helpful either on an abaxial or adaxial side by maintaining the transpiration rate76. An increase in density also maintains the water use efficiency under osmotic stresses74.

Conclusion

The present work focused on the effect of varying salt intensity and duration levels on the growth, physio-biochemical, and leaf microstructural architecture of Fimbristylis ecotypes under controlled environmental conditions. Both ecotypes of F. complanata showed a strong resilience to salt stress by displaying significant adaptation. The HR ecotype thrived better at a moderate saline regime (200 mM NaCl) with a short duration of stress exposure by enhancing growth and photosynthetic activity. This increase occurs due to the accumulation of photosynthetic pigment, which results in better photosynthesis performance. The key microstructural changes include enhancement in the aerenchyma area and midrib thickness for better water storage and transport of nutrients. The SH ecotype exhibited a greater salt tolerance in response to moderate to high salt (400 mM NaCl) conditions by improving the growth and photosynthetic efficiency. This ecotype maintained physio-biochemical and growth traits even when exposed to prolonged and higher salt levels. In conclusion, both ecotypes are well adapted, as the HR ecotype performed best at moderate salt stress while the SH ecotype excelled better under higher stress levels. The current findings suggest that F. complanata is a potential halophytic sedge for the phytoremediation of salt-affected lands and aquatic environments.