Freezing stress induces changes in the morphophysiological of chickpea and wild mustard seedlings

Wild mustard is one of the common and troublesome winter weeds of chickpea fields and a great competitor to reduce the chickpea productivity. Plant species (chickpeas cv. Saral and wild mustard) were compared at freezing temperatures (+4 as a control, 0, −4, −8, −12, −16, and −20°C) based on the morphophysiological traits and their recovery ability. Chickpea chlorophyll fluorescence was more sensitive to low temperatures than wild mustard. Chickpea and wild mustard Fv′/Fm′ (light‐adapted maximum efficiency of photosystem II [PSII] photochemistry) decrease 33% and 11% exposed to −16°C, respectively, compared with +4°C. Particularly at lower temperatures, wild mustard electrolyte leakage was smaller than that of chickpea; the temperature drop had a greater impact on the stems than the leaves. Per temperature degree drop from −12 to −20°C, the survival probability decreased by 12.5%. Wild mustard had a greater root dry matter (RDM) compared with chickpea plants. 50% dry matter depression temperature (RDMT50) could better distinguish among the species freezing response; wild mustard RDMT50 was ~1°C higher than chickpea. Plant survival and Fv′/Fm′ correlation suggested the reliability of chlorophyll fluorescence measurements to assay plants freezing tolerance. The important contribution of a more powerful root system to wild mustard survival under adverse circumstances may be suggested by the positive association between plant survival and RDM. Higher tolerance of wild mustard to freezing stress ultimately leads to greater survival, regeneration, continued growth, and geographical distribution. Therefore, the wild mustard invasion will be possible in chickpea fields after freezing stress, especially in the cold climates and high‐altitude regions.

temperatures than wild mustard. Chickpea and wild mustard F v 0 /F m 0 (light-adapted maximum efficiency of photosystem II [PSII] photochemistry) decrease 33% and 11% exposed to À16 C, respectively, compared with +4 C. Particularly at lower temperatures, wild mustard electrolyte leakage was smaller than that of chickpea; the temperature drop had a greater impact on the stems than the leaves. Per temperature degree drop from À12 to À20 C, the survival probability decreased by 12.5%.
Wild mustard had a greater root dry matter (RDM) compared with chickpea plants.
50% dry matter depression temperature (RDMT 50 ) could better distinguish among the species freezing response; wild mustard RDMT 50 was $1 C higher than chickpea.
Plant survival and F v 0 /F m 0 correlation suggested the reliability of chlorophyll fluorescence measurements to assay plants freezing tolerance. The important contribution of a more powerful root system to wild mustard survival under adverse circumstances may be suggested by the positive association between plant survival and RDM. Higher tolerance of wild mustard to freezing stress ultimately leads to greater survival, regeneration, continued growth, and geographical distribution. Therefore, the wild mustard invasion will be possible in chickpea fields after freezing stress, especially in the cold climates and high-altitude regions.  (Venkidasamy et al., 2019). Chickpea planting is widely popular in many parts of the world, especially in West Asia and North Africa, due to the low-input requirement. Chickpeas are grown in more than 50 countries worldwide, and India is known as the world's largest producer, with 73% (FAOSTAT, 2020). Iran, the United States, and Pakistan are among the largest producers of this product; each produces about 2% of the world's chickpeas (FAOSTAT, 2020). This plant is the main option in providing protein for the human body worldwide, especially in developing countries (Shafaei et al., 2016).
In Iran, with about 40% of production, chickpea is in the first place among pulse crops (Agricultural Statistics, 2018). According to Loke et al. (2016), the main reasons for consuming legumes are low fat and sodium and the lack of cholesterol and gluten in these products. Besides, because legumes are rich sources of iron, protein, fibers, and potassium, they can be used as important sources to meet the body's nutritional needs.
Weeds are one of the most critical factors that seriously reduce the chickpea yield in Iran on average by 50% (Nosratti et al., 2017).
Annual winter weeds can interfere directly with crops and serve as alternative hosts for important pests, especially in reduced tillage systems (Hayden et al., 2012). However, one of the crucial limitations of the successful production of chickpeas is the presence of weeds.
Due to dwarfism, slow establishment, and limited vegetative growth, chickpea plants have little ability to compete with weeds during the growing season (Toker et al., 2012).
Wild mustard (Sinapsis arvensis L.) is a broadleaf and annual winter weed that belongs to the Brassicaceae plant family. Wild mustard has indeterminate upright growth and may reach a height of more than two and a half meters (Siyahpoosh et al., 2012). This weed proliferates extreme spreading by producing thousands of seeds. Wild mustard is one of the most critical weeds in chickpea fields, distributed in most parts of Iran, and causes damage to autumn crops such as chickpeas (Shahbazi et al., 2019). Increasing the wild mustard density reduces the leaf area, canopy structure, plant height, and crop plant yield (Shahbazi et al., 2019). Besides, the stable seed bank, high competitiveness, high regeneration, and herbicide resistance are among the most critical problems of wild mustard control (Warwick et al., 2005), making it necessary to study it in various fields.
One of the most important factors that affected the growth and geographical distribution of plant species (crops and weeds) and cropweed interference is freezing stress. Plant tolerance to freezing stress is considered a factor determining their success after winter (Interrante et al., 2020;Nabati et al., 2021). On the other hand, examining the ability of weeds to compete with crops after the recovery period and adopting appropriate management methods to control them is considered one of the new strategies in weed control in autumn crops. Mittler (2002) defined "freezing" or "chilling" stress as damage to plant sensitive organs due to a sudden decline in temperature during the growing season, which can occur even at temperatures above 0 C (0 to 5 C). At this temperature range, the ice nucleus does not form in the cell.
After freezing stress, recovery capacity is one of the consequences of high tolerance to freezing stress in weeds, leading to more remarkable survival, continued growth, regeneration, and geographical distribution. Working on 12 weed species in different regions of Canada showed significant variations in freezing tolerance among the species. According to the experiment results, due to their high tolerance to cold stress, autumn weed species were more successful regarding their period and spatial distribution in the fields (Cici & Van Acker, 2011).
Freezing stress should be considered an important indicator in assessing plant suitability, including weeds and crops, in cold regions.
In other words, freezing stress tolerance is probably influential in the establishment and competitiveness of competing species. Therefore, according to the definition of fitness, that is, weed ability to establish, survive, and successfully regenerate under non-applying herbicides conditions, freezing tolerance is considered one component of fitness (Park et al., 2004). The high density of winter weeds such as wild mustard in spring in Iranian winter crops indicates the overwintering potential of these weeds . Therefore, the lack of proper cold acclimation of winter crops in future climate change scenarios increases the likelihood of winter weed dispersal and invasion.
Stomatal factors are considered one of the limiting factors of photosynthesis, which reduce carbon dioxide entrance into the intercellular space due to reducing stomatal conductance. This will disrupt the process of carbon fixation and photosynthesis (Ahmadi-Lahijani et al., 2018). Any environmental stress, biotic or abiotic, directly or indirectly, would affect the leaf stomatal aperture, which, in turn, interferes with the leaf photo assimilation rate. The plant cell membrane fluidity is decreased exposed to low temperatures, resulting in damage to the proteins and other membrane components. Besides, low temperatures reduce the activity of some enzymes, especially those involved in the photosynthetic procedure. The leaf chlorophyll fluorescence study can determine the value of damage to photosystem II (PSII) (Murchie & Lawson, 2013). Therefore, gas exchange variables and chlorophyll fluorescence will help to determine the plant stress tolerance.
In general, one of the determining factors in the geographical distribution and invasion ability of weeds is the threshold of freezing stress tolerance, the study of which, while improving basic information, will predict their distribution pattern and provide appropriate management strategies. Freezing and chilling stresses threaten chickpea fall cultivation. Wild mustard is one of the common winter weeds of chickpea fields and a great competitor to reduce chickpea productivity. Due to limited information on the competing ability of wild mustard with chickpeas under freezing stress conditions, this study was carried out aimed to (1) obtain basic information about the freezing tolerance threshold in wild mustard, (2) investigate the chlorophyll fluorescence and photosynthetic parameters of wild mustard and chickpea exposed to freezing stress, (3) compare the freezing tolerance of wild mustard compared to chickpeas to freezing stress, (4) assess the ability of wild mustard to compete with chickpeas after the recovery period, and (5) predict the distribution and possible invasion of wild mustard in cold regions according to its freezing tolerance threshold. 3 days at 5 C. The seeds were then placed in 9 cm Petri dishes on moist filter papers and kept at 20 C for 72 h to germinate. Both species seeds were sown in mid-November in plastic pots (12 cm diameter) containing farm soil, leaf mold, and sand (v:v 1:1:1) in a glasshouse (22/16 ± 2 C day/night and the photoperiod $10 h). Ten seeds of each plant species were sown in each pot separately and were tinned to five after establishment. The plants were normally irrigated during the growing periods. The last irrigation was done 24 h before the application of freezing treatments.
The pots were kept in natural conditions ( Figure 1) outside the glasshouse to the 2-4 leaf stage to adapt to the low temperatures for cold acclimation. Then, pots were moved to a thermogradient freezer to expose the freezing temperatures. First, the pots were placed at a temperature of 5 C. Then, the temperature decreased at a rate of 2 C per hour to reach the intended temperature. To prevent the ice nuclei formation, that is, the supercooling phenomenon, a thin layer of INAB (ice nucleation active bacteria) was sprayed on the seedlings to produce ice nuclei (Zhang & Liu, 2018). The plants were kept at the intended temperature for 1 h and then taken out of the thermogradient freezer and immediately placed in a growth chamber at 5 ± 1 C for 24 h to decrease the speed of ice melting. The pots were then placed in a pre-cold acclimation condition for 3 weeks to recover.

| Gas exchange variables
Leaf photosynthetic parameters were measured 1 week after freezing stress. The youngest fully developed leaves were used to measure the photosynthetic parameters between 10:00 a.m. and 2:00 p.m. three times for each treatment. Net photosynthetic rate (A N ), transpiration rate (E), and intercellular CO 2 concentration (C i ) were measured using a portable photosynthesis system (ADC Bio Scientific Ltd, UK) at approximately 1000 μmol m À2 Ás À1 PAR (photosynthetically active radiation), relative humidity of 40% ± 5%, the ambient CO 2 concentration, and a 25 C leaf temperature. Stomatal conductance (g s ) was measured using a portable leaf porometer (SC-1, USA). Instantaneous (WUE i ) and intrinsic (A N /g s ) water use efficiency were calculated by dividing A N by E and g s , respectively. Mesophyll conductance (g m ) was also calculated by dividing A N by C i (Ahmadi-Lahijani & Emam, 2016).
At the same time, leaf chlorophyll content (SPAD value) was measured on the same leaves using a handheld chlorophyll meter (SPAD 502, Spectrum Technologies, Inc.).

| Leaf chlorophyll fluorescence
Leaf chlorophyll fluorescence parameters, including light-adapted maximum efficiency of PSII photochemistry (F v 0 /F m 0 ), photochemical quenching (qP, also known as F q 0 /F v 0 ), and the quantum efficiency of PSII electron transport (ΦPSII, also known as F q 0 /F m 0 ), were recorded from the youngest fully developed leaf by a fluorometer (Opti Science, Inc.). The glass fiber was set at a 1 mm distance from the leaf and a saturation pulse PPFD (photosynthetic photon flux density) at $7500 μmolÁm À2 Ás À1 for 0.8 s. Chlorophyll fluorescence was measured before transferring the plants to the freezing temperatures.
The trend of chlorophyll fluorescence changes was recorded 6, 12, 24, 48, and 72 h after freezing stress (AFS) during a recovery period.
The linear electron transport rate (J) described as in Equation (1) (Genty et al., 1989): F I G U R E 1 Maximum, minimum, and mean air temperatures during the experiment PFDa (absorbed photon flux density) is the absorbed light (μmolÁm À2 Ás À1 ), and 0.5 is a factor (accounts for the partitioning of energy between PSII and PSI). It is generally not practical to measure the light absorbed by a leaf; therefore, relative changes of J can usefully be monitored by simply multiplying ΦPSII by incident light (Maxwell & Johnson, 2000). Therefore, Equation (2) can be modified as

| Electrolyte leakage (EL)
A complete plant with two fully developed true leaves and its stems was separated and placed in vials containing 50 ml of deionized water from each pot. The vials were placed in the laboratory temperature for 24 h, after which the electrolyte leakage was measured using an electrical conductivity meter (Jenway Model 4510) and recorded as EC 1 . The vials were transferred to an autoclave (110 C and 1.2 atm) for 30 min. They were then placed in a laboratory temperature for 24 h, and the electrical conductivity was again measured and considered as EC 2 . The percentage of electrolyte leakage (EL%) was calculated using Equation (3) (Ghoulam et al., 2002).

| Plant survival (SU)
To determine the survival percentage and plant regrowth, the pots were transferred to the greenhouse at $20 C and kept for 21 d under a natural photoperiod. The percentage of plant survival was measured by counting the number of alive plants before frost stress and 21 days after the freezing stress using Equation (4).
Here, B and A are the number of alive plants before and after freezing stress, respectively.
2.2.6 | 50% lethal temperature (LT 50 ) To determine the lethal temperature of 50% of the plants based on the 50% dry matter depression temperature (RDMT 50 ) and the survival percentage (LT50 su ), the logistic equation for the data of each species at different temperatures was fitted using Equations (5) and (6), respectively (Eizenberg et al., 2005): In this equation, y represents the survival percentage, x represents the freezing temperature, a is one of the coefficients of the equation and represents the maximum survival percentage, b is another coefficient of the equation and represents the slope of the curve at point x, and xc represents the point of x at which y is equal to 50% of its maximum value (LT 50  3 | RESULTS

| Leaf chlorophyll content (SPAD value)
According to Figure 2a, the SPAD value decreased with a sharp slope after a stable trend up to À12 C and ultimately reached zero at À20 C. Wild mustard SPAD was higher at all temperatures compared with chickpea plants. Decreasing temperature to À16 C decreased SPAD by 4.8 and 4.6 times in chickpea and wild mustard, respectively, compared with À12 C (Figure 2a).

| Electrolyte leakage (EL)
The damage to cell membranes was significantly affected by temperature and plant species (Figure 2). With decreasing temperature, the EL percentage increased, in which chickpea plants were more susceptible than wild mustard; that is, wild mustard EL was lower than that of chickpea in most temperatures. Stems were more affected by the temperature decline; for example, the percentages of chickpea and wild mustard EL stem were 88% and 75% at À20 C, respectively, whereas the EL leaf was 70% and 60% chickpea and wild mustard, respectively, at À20 C (Figure 2b,c). Up to À12 C, there was a relatively constant trend in leaf and stem EL of the species; however, it significantly increased at À16 C thereafter.

| Plant survival
Freezing temperatures had significant impacts on the survival percentage of plant species. A decline in temperature from 0 to À12 C did not affect the plant SU, but it showed a sharp decline after À12 C ( Figure 2d); that is, survival percentage decreased by 12.5% each temperature degree depression from À12 to À20 C.

| Gas exchange variables
Gas exchange variables of both plant species statistically remained unaltered to À12 C, but À16 C dramatically decreased those parameters compared with the higher temperatures. Wild mustard showed higher A N before the stress onset than chickpea at all temperatures; however, it was more affected by freezing temperatures (Table 1).
With decreasing the freezing temperatures, A N was declined in both plant species. Up to À12 C, no significant decrease was observed in the plant species A N compared with temperature 4 C; but decreasing temperature to À16 C diminished chickpea and wild mustard A N 1.3 and 1.5 times, respectively, compared with 4 C. Nevertheless, both plants A N reached zero at À20 C. The same trend was observed for g s ; for instance, chickpea and wild mustard g s were decreased by 1.3 and 3.3 times, respectively, compared with 4 C. Although chickpea WUE i decreased by declining temperature to À12 C compared with before stress, at À16 C, WUE i showed a higher value compared with the higher temperatures. Wild mustard had a higher WUE i at all temperatures compared with chickpea (Table 1).

| Leaf chlorophyll fluorescence
Chlorophyll-a fluorescence was recorded just before the onset of and after 6, 12, 24, and 48, and 72 h after freezing stress (AFS) at different temperatures ( Figure 3). Generally, chickpea leaf chlorophyll fluorescence was more sensitive to low temperatures compared with that of wild mustard. The maximum light-adapted quantum yield of PSII photochemistry, F v 0 /F m 0 , was recorded at 4 C (Figure 3a,b).
Chickpea F v 0 /F m 0 exposed to À16 and À20 C decrease 33% and 43%, respectively, compared with 4 C and continued to decrease to reach zero at 48 h. Whereas in wild mustard, F v 0 /F m 0 decreased only by 11% at both À16 and À20 C compared with 4 C and reached zero at 72 h.
F I G U R E 2 Changes in leaf chlorophyll content (SPAD) (a), leaf (b) and stem electrolyte leakage (c), and survival percentage (d) of chickpea and wild mustard plants under freezing temperatures. Each point is average, and vertical bars indicate ±SD (n = 4). Asterisk represents significant differences between the plants at p ≤ 0.05.
T A B L E 1 Photosynthetic variables of chickpea and wild mustard plants before and after exposure to freezing temperatures   The maximum quantum yield of PSII, ΦPSII, was affected differently in the plant species. Chickpea ΦPSII decreased 33% and 43% in À16 and À20 C treated plants, respectively, 6 h AFS compared with 4 C. Nevertheless, wild mustard ΦPSII only decreased by 10% at both À16 and À20 C 6 h AFS compared with 4 C (Figure 3c,d). Leaf photochemical quenching showed the same trend in both species, and it was not affected by temperatures to À16 C; however, qP showed different behaviors in plants exposed to À20 C.
Wild mustard qP started to decrease with a sharp slop 48 h AFS, but

| Plant growth parameters
Plant species and temperature interacted to affect the growth parameters. Although chickpea plants had a greater SHDM than wild mustard, a decrease in temperature from zero to À12 C reduced chickpea SHDM by 31% compared with À8 C, whereas wild mustard SHDM remained unaltered to À12 C. However, a further decrease in temperature to À16 C decreased wild mustard SHDM as well, but with a gentler slope (Figure 4a). RDM showed a contrariwise behavior; wild mustard had a greater RDM compared with chickpea plants, but the same sensitivity trend was observed as for SHDM (Figure 4b).
However, both plant species SHDM and RDM reached zero at À20 C.
Both species LA showed a similar trend. No significant LA decrease was observed to À12 C, although wild mustard had a greater LA than chickpeas (Figure 4c). LA per plant decreased 68% and 130% in chickpea and wild mustard, respectively, at À16 C compared with À12 C. Chickpea plants, on the other hand, had a greater LN per plant than wild mustard but with a higher sensitivity to low temperatures (Figure 4d). There were no changes in wild mustard LN to À12 C, whereas chickpea LN decreased by 37% by a 4 C temperature decline from À8 to À12 C. Plant height showed a similar trend in both plant species. To À12 C, no decline was observed in neither plant species; however, further temperature decreases to À16 C decreased chickpea plant height by 70% compared with À12 C (Figure 4e). Chickpea plants had greater branches than wild mustard; wild mustard plants had no branches (single stem) that remained to À20 C. Chickpea branch number decreased with a gentle slope by temperature decline and reached zero at À20 C (Figure 4f).

| LT 50su and RDMT 50
Both species remained LT 50su up to À12 C, but the SU was declined by increasing the freezing temperature intensity (Figure 5a). Chickpea lost 50% of plant survival at À16.1 C, whereas wild mustard lost 50% of plant survival at À15.9 C. A significant difference (1.01 C) was observed between the plant species in terms of RDMT 50 . Chickpea with an RDMT 50 of À15.7 C showed a lower rate of regrowth after F I G U R E 4 Changes in the shoot (a) and root dry matter (b), leaf area (c), leaf number (d), plant height (e), and branch number (f) of chickpea and wild mustard plants under freezing temperatures. Each point is average, and vertical bars indicate ±SD (n = 4). Asterisk represents significant differences between the plants at p ≤ 0.05.
F I G U R E 5 50% lethal temperature based on survival percentage (LT 50su ) (a) and 50% dry matter depression temperature (RDMT 50 ) (b) of chickpea and wild mustard plants under freezing temperatures the recovery period compared with wild mustard, which means that in chickpea, a decrease in temperature to less than À15.7 C caused a 50% decrease in DM (dry matter) at the end of the recovery period, whereas decreasing the temperature to less than À16.7 C reduced wild mustard DM by 50% (Figure 5b).

| DISCUSSION
The freezing tolerance of a plant varies significantly among different tissues. For instance, stem, meristems, the lower and upper leaves of the plant canopy, and roots have different freezing tolerance (Herzog, 1987;Herzog & Olszewski, 1998). Antifreeze proteins and ice nuclei control the initial formation of ice. Such mechanisms as membrane fluidity and osmotic regulation are often associated with freezing tolerance at the cellular level. Both metabolic and physiological alterations in the plant in response to low temperatures highly affect the cold acclimation or hardening process. Alteration in cellular and metabolic status, including greater sugars, soluble proteins, proline, organic acids, and altered lipid membrane composition, may lead to cold acclimation (Hughes & Dunn, 1990. Plant DM decreased as temperature declined in both species. The results indicated that both leaf and stem EL were increased, but the enhancement of EL and plant DM depletion was greater in chickpea plants than wild mustard. A negative correlation was observed between the EL with survival and plant DM. Previous findings also indicated the adverse effect of freezing stress on cell membrane stability (Bertin et al., 1996;Kaur et al., 2008). The freezing stress tolerance evaluation of Trifolium hirtum showed that the leaf EL increased with declining temperature from À6 to À14 C (Nunes & Smith, 2003).
An investigation on the effect of cold stress on two wheat (Triticum aestivum) cultivars at the seedling stage showed that ion leakage levels were not affected by temperatures above 0 C, whereas EL of plants was increased with decreasing temperatures below zero (Apostolova et al., 2008). However, in the present study, it was observed that even temperatures below 0 C did not affect the leaf EL in wild mustard to À12 C, a sign of greater tolerance of wild mustard.
High EL values indicate the membrane's lack of ability to retain intracellular compounds; more electrolytes leak from the membrane and damage the cell membrane. Studies showed that the unsaturated fatty acids present in cell membranes are essential in membrane fluidity.
Low temperatures change the fluidity of these membrane fatty acids from semi-liquid to crystalline (Mahajan & Tuteja, 2005), and subsequently, ionic leakage increases. Reactive oxygen species (ROS) produced under low temperatures can react with the membrane lipids and cause lipid peroxidation, leading to cellular content leakage and rapid cell dehydration and cell death (Takac, 2004). These changes give rise to other effects of chilling or freezing on the plant and cell levels (Blum, 2018).
Although differently, freezing stress decreased the F v 0 /F m 0 of both plant species; chickpea plants showed more sensitivity than wild mustard. The leaf F v 0 /F m 0 of chickpea plants exposed to À16 C significantly decreased 6 h AFS and reached zero after 48 h of the recovery period. Accordingly, ΦPSII and J also showed a similar trend. None of the plant species recovered their fluorescence parameters during the recovery period. Hasanfard et al. (2021) indicated that the leaf F v 0 /F m 0 decreased in turnipweed (Rapistrum rugosum (L.) All.) with a decrease in temperature from À12 C and during the first 24 h after the freezing treatment; the F 0 v /F 0 m levels decreased by 28% compared to before the freezing stress. They found that a temperature decline from À12 C disrupted the carbon exchange and PSII electron transport. Chlorophyll fluorescence measurement is a reliable and appropriate tool to evaluate plant tolerance to low temperatures (Ehlert & Hincha, 2008;Rizza et al., 2001). This method can reveal the susceptibility of the PSII electron transport chain (Maxwell & Johnson, 2000) and provide a non-destructive and faster diagnostic tool for evaluating the effect of freezing stress on plants than the destructive methods such as the EL (Christen et al., 2007;Su et al., 2015).
Plants are more sensitive to low temperatures in the autotrophic than the heterotrophic stage. Freezing temperatures mainly impact the growing seedlings by the cell membrane damage and cause the respiration and photosynthesis to decrease. Besides, plant wilting due to loss of leaf turgor results in temperature-induced drought stress. In the present study, temperatures below À12 C significantly reduced A N and photosynthetic variables, likely due to the cell membrane damage and electron transport chain disruption. Photosynthesis is regulated by stomatal and non-stomatal factors, depending on plant species and the environmental conditions (Ahmadi-Lahijani et al., 2018). Freezing and chilling temperatures induce water loss through a slow stomatal aperture, increased membrane permeability, lower root hydraulic conductivity, and root water uptake (McWilliam et al., 1982;Wolk & Herner, 1982).
Wild mustard A N /g s showed an increasing trend by decreasing the temperature up to À16 C, with the greatest value at À16 C ( Figure 6b). The species behaved differently; freezing-stressed chickpeas had relatively lower A N /g s compared with the unstressed plants, whereas it was vice versa in wild mustard, relatively higher A N /g s in freezing-stressed plants compared with the unstressed (Figure 6a,b). It may indicate that lower chickpea A N was due to a greater stomatal limitation under relatively lower temperatures. Plants vary in their capacity to regulate how much water they lose per unit carbon gained, determining by "intrinsic WUE," A N /g s (Condon et al., 2002). Although wild mustard is a C 3 species, its extensive root system and large photosynthetic capacity make it a very competitive weed (Szmigielski et al., 2015). Besides, wild mustard's higher stomatal density than many other broadleaves weed species leads to a higher photosynthetic rate, faster plant (re)growth, and lower stomatal closure effect on the leaf photosynthesis. However, the greater decrease in wild mustard C i :C a by decreasing temperature (e.g., À16 C) indicating the greater role of stomatal factors in photosynthetic regulation even at extremely low temperatures (Figure 6d).
A N was significantly diminished in both species at À16 C, indicating the damages imposed to photosynthetic apparatus at low temperatures. Wild mustard A N was higher compared with chickpea up to À16 C. This, along with a relatively lower E, led to a rise in wild mustard WUE i compared with chickpea. Water use efficiency is considered an important factor in crop yield determination and one of the parameters to assay plant freezing tolerance (Navarrete-Campos et al., 2013). Low temperature (À5 C for 12 h) reduced tea (Camellia sinensis L.) plantlet g s by 88% compared with the control (Li et al., 2018). Gupta et al. (2016) also found a decrease in g s in plants exposed to various low temperatures. Positive correlations were observed between A N with g s and g m (Figure 7), suggesting that these variables were coupled. Chilling and freezing stress reduces the conductivity of the tonoplast and plasmalemma of the guard cells, resulting in the stomata less responsive to leaf water potential alterations in sensitive species. Under conditions of continued evaporative demand in the light, slow closure of stomata and reduced water uptake reduce water potential, leading to tissue dehydration (McWilliam et al., 1982).
Chickpea SPAD value started to decline with a gentle slope after a stable trend at À8 C, whereas wild mustard SPAD value did not change to À12 C compared with +4 C. However, both species experienced a sharp decrease afterward. Cold-acclimated rapeseed plants showed a higher SPAD value by 41% than the unacclimated plants (Nezami et al., 2009). SPAD and A N were positively correlated ( Figure 6), indicating the close relationship between leaf pigment content and photosynthetic activities.
Plant survival after freezing stress in crops and their common weeds sheds more light on the dynamics of their distribution and competitiveness in areas with low temperatures. Plant survival remained unaffected to À12 C in both species; however, reducing the temperature to À16 C decreased the plant SU. The exponential set curve showed that both species survived above 50% to $À16 C ( Figure 5a). However, all plants died at the temperature of À20 C. A highly positive correlation coefficient (r = +0.97 ** ) was observed between the plant survival percentage and A N (Figure 7). The linear regression between plant SU and A N suggested that the survival rate increased with increasing the rate of A N . LT 50su has been reported as a suitable indicator of cold tolerance in plants (Fowler et al., 1996;Liang et al., 2003). Nezami et al. (2016) observed that 50% of chickpea plant lethal temperature was about 6 C lower in the tolerant than the susceptible species. Wery (1990) found that although some genotypes tolerate À12 C in the vegetative stage after emergence, the minimum temperature at which chickpeas generally survive is À8 C. Increasing autumn temperatures resulting from climate change in Iran led to reduced freezing tolerance of autumn crops .
In other words, due to the lack of optimal cold acclimation in autumn, chickpeas will not tolerate freezing stress. As a result, they will be damaged by decreasing temperatures during winter. However, weeds generally have a higher ability to tolerate freezing stress (Cici & Van Acker, 2011). Based on this, it can be inferred that climate change is adversely shifting weed flora in an Iranian cropping system.
As temperatures declined below À12 C, both species suffered severe damage; at À16 C, the LA was dramatically diminished due to plant death. A similar study found that turnipweed LA decreased less at À12 C compared with that of wild oat (Avena ludoviciana Durieu.) . Higher A N was correlated with a greater SHDM (Figure 7). Cold acclimation requires the energy supplied by photosynthetic activities. However, during cold acclimation, the chloroplast properties are changed (Huner et al., 1998). Experiments to assay freezing tolerance of chickpea and grass species revealed that plant dry matter was decreased by temperature decline to À8 C Nezami et al., 2016). Plant growth reduction might associate with a slower rate of food reserve transfer and reduced photoassimilate mobilization due to reduced enzyme activity (Powell & Matthews, 1978).
Wild mustard had a greater RDM than chickpea, which remained longer and tolerated freezing temperature to À12 C without any F I G U R E 6 Changes in the net photosynthesis rate to stomatal conductance ratio in chickpea (a) and wild mustard (b), and substomatal to ambient CO 2 concentration ratio in chickpea (c), and wild mustard (d) plants under freezing temperatures. BS, before stress; AS, after stress. Each point is average, and vertical bars indicate ±SD (n = 4). Asterisk represents significant differences between the time of measurement at p ≤ 0.05. significant changes. However, wild mustard RDM started to reduce in plants exposed to À16 C; nevertheless, it was still greater than chickpea RDM. The greater survival of wild mustard might be due to the greater RDM. A highly positive correlation between plant SU and RDM (r = +0.96 ** ) (Figure 7) may indicate the bold role of a more robust root system in plant survival under stressful conditions. Weeds have always been a strong competitor to crops for environmental resources; the greater underground parts of weeds might be their secret of better survival.
Studies showed that sometimes freezing stress may not kill some plants, although it affects plant regrowth during recovery. Therefore, plants might not fully recover and resume their regrowth properly under such conditions. The index RDMT 50 can distinguish between the plants for freezing tolerance and provide more precious discrimination. In the present study, the plant species differed in RDMT 50 .
Although there was no significant difference in LT 50su of plant species, wild mustard showed a better regrowth with a higher RDMT 50 (À15.6 vs. À16.6 C in chickpea and wild mustard, respectively) during the recovery period (Figure 5b). Plant dry matter is related to physiological responses and decreases during the recovery period due to freezing damage to plant physiological processes and regrowth ability (Azizi et al., 2007). Izadi Darbandi et al. (2020) also reported that the seedlings of wild barley (Hordeum spontaneum Koch.) and feral rye (Secale cereale L.) with RDMT 50 of À8.1 and À11.6 C, respectively, were the most susceptible and most tolerant plants under freezing stress. Nezami et al. (2007) observed a high correlation between LT 50 and RDMT 50 in chickpea genotypes. They found RDMT 50 was lower in the tolerant than susceptible genotypes. Hekneby et al. (2006) also found that forage legume tolerant species had better regrowth than susceptible species. F I G U R E 7 Pearson's correlation between the experimental parameters in chickpea and wild mustard plants under freezing temperatures. A N , net photosynthetic rate; C i , substomatal CO 2 concentration; E, transpiration rate; EL, electrolyte leakage; F v 0 /F m 0 , light-adapted maximum efficiency of PSII; g m , mesophyll conductance; g s , stomatal conductance; RDW, root dry weight; ShDW, shoot dry weight; SPAD, leaf chlorophyll content; SU, survival percentage; WUE, water use efficiency

| CONCLUSIONS
Due to the increasing cultivation area of autumn chickpea in Iran, it is predicted that wild mustard interruption, especially in cold regions, will increase in the future. Plant species show various levels of tolerance and sensitivity to environmental stresses. Therefore, evaluating the freezing tolerance of wild mustard helps us better understand how this weed will distribute and invade. The significant correlation between F v 0 /F m 0 and plant survival suggested the reliability of chlorophyll fluorescence measurements to assay plant freezing tolerance. The results showed that temperatures below À12 C decreased both species' photosynthetic variables. Freezing stress reduced plant survival percentage and the shoot dry matter at the end of the recovery period, although the species response varied depending on the intensity of stress. Although LT 50su of plant species did not significantly differ, RDMT 50 could distinguish between the species freezing response; wild mustard RDMT 50 was $1 C higher than chickpea.
Accordingly, chickpea showed more sensitivity to freezing stress than wild mustard in this experiment. The positive correlation between plant survival and root dry matter may indicate the significant role of a more robust root system in wild mustard survival under stressful conditions. Overall, due to climate change and temperature fluctuations in winter, it seems that wild mustard has a high ability to adapt to these conditions and, if not controlled, can lead to more damage, especially in Iranian chickpea fields. Obtained data from this study can be used in other similar climates and other cropping systems. Our findings predict that moving away from winter weed management, such as lack of crop rotation, will encourage wild mustard dispersal under harsh winter conditions.

ACKNOWLEDGMENT
We are grateful to the Ferdowsi University of Mashhad, Iran, for providing financial support for present research (Project No. 2.51336).