Stable expression of aquaporins and hypoxia-responsive genes in adventitious roots are linked to maintaining hydraulic conductance in tobacco (Nicotiana tabacum) exposed to root hypoxia

Formation of adventitious roots in plants is a common response to hypoxia caused by flooding. In tobacco, after one week of root hypoxia treatment, plants produced twice as many adventitious roots as the aerated plants, but their maximum length was reduced. Hypoxia severely reduced net photosynthesis, transpiration rates, and photosynthetic light responses. Relative transcript abundance of the examined aquaporins in lateral roots was reduced by hypoxia, but in adventitious roots it remained unchanged. This apparent lack of an effect of root hypoxia on the aquaporin expression likely contributed to maintenance of high hydraulic conductance in adventitious roots. Lateral roots had lower porosity compared with adventitious roots and the expression of the ACS (1-aminocyclopropane-1-carboxylate synthase) gene was induced in hypoxic lateral roots, but not in adventitious roots, providing additional evidence that lateral roots were more affected by hypoxia compared with adventitious roots. ATP concentrations were markedly lower in both hypoxic lateral and adventitious roots compared with aerated roots, while the expression of fermentation-related genes, ADH1 (alcohol dehydrogenase 1) and PDC1 (pyruvate decarboxylase 1), was higher in lateral roots compared with adventitious roots. Since root porosity was greater in adventitious compared with lateral roots, the results suggest that the improved O2 delivery and stable root aquaporin expression in adventitious roots were likely the key factors helping flooded tobacco plants maintain high rates of root hydraulic conductance and, consequently, shoot gas exchange.


Introduction
Frequency of floods is predicted to increase globally due to the climate changes [1]. As O 2 diffusion rate is extremely low in water compared to air, prolonged flooding can lead to O 2 deprivation and reductions of growth and survival in terrestrial plant species [2]. Root O 2 deficiency (hypoxia) limits respirational ATP synthesis and results in an energy crisis and toxicity PLOS  study, hydroponically grown tobacco plants were subjected to root hypoxia to shed more light on the processes in hypoxia-induced ARs that facilitate water transport. It was hypothesized that root hydraulics and ATP production under hypoxia can be enhanced by the formation of ARs. Root porosity, ATP contents and transcript profiles of hypoxia-responsive genes were compared between ARs and existing LRs. Transcription profiling of PIPs in ARs was also compared to LRs to examine potential significance of various PIPs in the responses of roots to hypoxia.

Growth conditions and hypoxia treatment
Tobacco seeds were germinated and seedlings were grown in horticultural soil a controlled growth room with 18 h photoperiod, 22/18˚C (day/night) temperature, 400 μmol m -2 s -1 photosynthetic photon flux density, 350 μmol mol −1 CO 2 and 50% relative humidity. After 3 weeks of growth, plants were transferred to 40-L plastic tubs (~60×40×20 cm) containing 50% strength modified aerated Hoagland's solution [45]. Thirty-two plants were randomly selected and grown in four tubs (8 plants in each tub). After one week, 16 plants in two tubs were subjected to hypoxia by flushing nitrogen gas (99.998%, Praxair, Danbury, CT, USA) through the solution to reach a dissolved O 2 level of~2 mg L -1 and then leaving the solution stagnant. The other 16 plants in two tubs were well-aerated with air pumps and served as control (dissolved O 2 concentration of~8 mg L -1 ).

Gas exchange and photosynthetic light responses
After two days and one week of treatments, gas exchange was measured with the Li-Cor LI-6400XT portable photosynthesis system equipped with the 2×3 cm 2 red-blue light chamber (Li-Cor, Lincoln, NE, USA). Six plants in each treatment and three middle-position fully expanded leaves on each plant were randomly selected. Net photosynthetic rate (P n ), transpiration (E) and stomatal conductance (g s ) were measured. Air flow rate was set to 400 μmol s -1 , photosynthetic photon flux density (PPFD) was 400 μmol m -2 s -1 , and reference CO 2 concentration was 400 μmol mol -1 . An automated program of LI-6400XT was used to determine photosynthetic light responses starting at PPFD of 1500 μmol m -2 s -1 , followed by 1200, 1000, 800, 500, 300, 200, 100, 50, 20 and 0 μmol m -2 s -1 . Three plants in each treatment were randomly selected and net photosynthesis was auto-logged when it reached a steady rate. A modified rectangular hyperbole model was employed to estimate light saturated P n (P m ), light saturation point (I m ) and light compensation point (I c ) [46].

Root hydraulic conductance (K r )
A high-pressure flow meter (HPFM, Dynamax Incorporated, Houston, TX, USA) was used to measure tobacco root K r as previously described [47,48]. Shoots were excised about 2 cm above the root collar, and the roots were connected to the HPFM. Roots were kept in treatment solutions during the measurements. Water was forced into roots at increasing pressures (0 to 0.5 MPa) and linear regression between applied pressure and flow rate was used to obtain a slope of the relationship which represented K r .

Number, dry mass and maximum length of ARs
After one week of treatment, number of ARs was counted and the maximum length of ARs was measured with a ruler. Dry mass of ARs was determined after drying the roots in an oven at 85˚C.

Root porosity
Root porosity in lateral (LR) and adventitious (AR) roots was estimated with the pycnometer method based on Archimedes' principle [49] after one week of treatment using the following equation: where M w is mass of the water filled pycnometer, M r is mass of roots, M r+w is mass of pycnometer with roots and water and M h is mass of pycnometer with homogenized roots [49].

Root ATP concentration
LRs and ARs were sampled and ground in liquid nitrogen after one week of hypoxia treatment. ATP concentration was determined with the ENLITEN ATP Assay Kit (Promega, Madison, WI, USA) by measuring bioluminescence and quantified with a standard curve using ATP standard (Promega). Luminescence signal was detected using a microplate reader (Fluostar Optima, BMG Labtech, Ortenberg, Germany) as previously described [28].

RNA transcription profiling
After two days and one week of treatment, LRs and ARs from 6 plants in each treatment were sampled. The samples were quickly frozen and kept in liquid nitrogen before being transferred to the -80˚C freezer. The samples were ground with a mortar and pestle in liquid nitrogen. Total RNA was extracted with a Plant RNeasy extraction kit (Qiagen, Valencia, CA USA). First strand of cDNA was synthesized from 1μg total RNA using a Reverse Transcription Kit (Qiagen). Quantitative RT-PCR was employed to analyze relative RNA expression using the 2 -ΔCt method. The relative transcript abundance of PIPs was normalized against geometric mean of the CT value of two reference genes, NtEF1-α (AF120093) and L25 (L18908) [50]. Transcripts levels of NtPIP1;1 (AF440271), PIP1;2 (= AQP1, AF024511), PIP1;3 (U62280), PIP1;4 (DQ914525) and PIP2;1 (AF440272) were analyzed. These aquaporins were selected because of their sequence availability in the public database and earlier studies showing their functional importance [25,28]. NtADH1 (alcohol dehydrogenase 1, X81853.1) and PDC1 (pyruvate decarboxylase 1, X81854.1) were selected as hypoxic indicators. Relative transcript abundance of ACS (X65982.1) was also determined since it encodes an enzyme catalyzing a rate-limiting step in ethylene synthesis [51]. Gene-specific primers are described in S1 Table.

Statistical analysis
Means (n = 3-6) and standard errors (SE) were calculated. Paired t-test was performed to compare AR formation and K r between aerated and hypoxic roots (α = 0.05). In all other comparisons, one-way ANOVA followed by Tukey's test was performed to compare means (α = 0.05). Three out of four replications of PDC1 relative transcript abundance of aerated LRs were too low to be detected and consequently only the relative transcript abundance of PDC1 of aerated ARs, hypoxic LRs and hypoxic ARs were compared in Tukey's test.

Leaf gas exchange and photosynthetic light responses
After one week of treatment, the leaves of hypoxic plants showed no signs of chlorosis or wilting. P n of hypoxic plants decreased by over 50% and 70% compared with aerated plants after two days and one week of treatment, respectively ( Fig 1A). Hypoxic plants also showed a decrease of more than 70% in E and g s on the two treatment days (Fig 1B and 1C). Hypoxia treatment profoundly affected photosynthetic light responses ( Fig 1D). Both two days and one week of hypoxia treatments resulted in significant declines of I c and I m ( Table 1). P m of hypoxic plants also showed a significant decrease compared with aerated plants after one week of treatment (Table 1).

AR formation and root porosity
After one week of treatment, root mortality was observed in the lower part of hypoxic roots. Formation of ARs was induced by hypoxia around the stem base (Table 2). Hypoxic plants had over two-fold higher AR number compared with aerated plants. However, the maximum length of ARs in hypoxic plants was significantly lower compared with aerated plants ( Table 2). Hypoxia treatment did not affect the total dry mass of ARs (Table 2).

Fig 1. Net photosynthesis (P n , A), transpiration rates (E, B), stomatal conductance (g s , C) and photosynthetic light responses (D) of well-aerated tobacco plants and plants subjected to root hypoxia treatment for two days and one week.
Means ± SE (n = 5 or 6 for P n , E and g s . and n = 3 for photosynthetic light responses) are shown. Different letters above the bars indicate statistically significant differences determined by the Tukey's test after one-way ANOVA (P � 0.05). Aerated after two days (open triangles), hypoxia after two days (open circles), aerated after one week (black triangles), and hypoxia after one week (black circles).
https://doi.org/10.1371/journal.pone.0212059.g001  Root air space was estimated by the root porosity test after one week of treatment. Hypoxia lead to a 13% increase of porosity in ARs and a slight decrease of porosity in LRs (Fig 2A). The porosity of hypoxic ARs was higher by over 50% compared with hypoxic LRs. The porosity of aerated ARs was higher by over 25% compared with aerated LRs.

K r and root ATP concentration
There was no significant difference in K r between hypoxic roots and aerated roots after one week of treatment ( Fig 2B).
After two days of treatment, hypoxia resulted in a significant decrease of ATP concentration in both ARs and LRs (Fig 2C). ATP concentration of hypoxic LRs and ARs decreased by about 60% and 40%, respectively, compared with aerated roots (Fig 2C). After one week of treatment, no significant difference in ATP concentration between hypoxic and aerated roots was detected.

RNA expression profiling in roots
Hypoxia resulted in significant decreases of PIP1;1 and PIP1;3 relative transcript abundance in LRs after two days of treatment ( Fig 3A). However, relative transcript abundance in ARs remained unchanged ( Fig 3A). Relative transcript abundance of ACS, ADH1 and PDC1 in LRs were sharply induced by hypoxia (Fig 3B). Hypoxic ARs also showed significantly higher ADH1 relative transcript abundance compared with aerated ARs, however, ACS remained unchanged (Fig 3B). After one week of treatment, relative transcript abundance of PIP1;2, PIP1;4 and PIP2;1 in hypoxic LRs were significantly lower compared with the aerated LRs, whereas relative transcript abundance of PIP1;1 and PIP1;3 showed no change (Fig 3C). In contrast, hypoxia did not result in significant changes of PIP relative transcript abundance in ARs (Fig 3C). Relative transcript abundance of ACS, ADH1 and PDC1 exhibited similar trends as on day two ( Fig  3D). A sharp increase of ACS, ADH1 and PDC1 expression was triggered in LRs by hypoxia (Fig 3D). Hypoxic ARs showed no changes in relative transcript abundance of the ACS, ADH1 and PDC1 compared with aerated ARs (Fig 3D).

Discussion
O 2 deficiency is a challenging environmental factor that produces complex responses in plants.
Following two days and one week of treatment, gas exchange in tobacco was sharply reduced by hypoxia. Hypoxia also increased the number of ARs but with a similar biomass as in aerated plants. ARs showed different response patterns to hypoxia compared with the LRs in terms of the transcript profiles of PIPs and hypoxia-responsive genes, which may partially contribute to maintaining K r of hydroponically-grown tobacco plants.
Stomatal conductance, which is regulated by hydraulic and (or) chemical signals, is the main limiting factor for leaf carbon assimilation [52]. The decline in P n after two days and one week of hypoxia was likely due to the stomatal closure, which was reflected by the decreased E. The decrease in stomatal opening, as demonstrated by lower g s , was likely responsible for the reductions in photosynthetic light responses, which showed that P n of hypoxic plants was saturated at a significantly lower light intensity. Reduced g s of hypoxic leaves limits internal CO 2 concentration used for carbon assimilation. Root hypoxia also reduced I c , the light intensity at which the rate of total photosynthesis is balanced by the rate of respiration suggesting that root hypoxia inhibited leaf respiration in addition to photosynthesis. However, hypoxic plants maintained positive P n at similar rates on days two and seven, which demonstrates relative tolerance of tobacco plants of hypoxic conditions. One week of root hypoxia did not influence K r in hydroponically grown tobacco. Effects of O 2 deprivation on root hydraulics varies between plant species and experimental conditions. While some studies reported reduced K r in response to O 2 deficiency [53,54], other studies showed no effect [44,55]. In contrast to K r , hypoxia resulted in a significant decrease of E in the present study. E and K r are frequently strongly linked in plants [56], but this relationship may also be affected by other factors including leaf to root ratio [57]. Root growth is typically reduced more than leaf growth under hypoxic conditions [58]. In the present study, leaves of hypoxic plants showed no chlorosis and wilting, but root mortality was observed. Thus, even though K r remained unchanged under hypoxic conditions, the increasing transpiration demand, as a result of leaf growth, may still lead to decreased E in the present study. The diurnal changes of root hydraulic conductivity were also found to be independent of transpiration in flooded Zea mays [59]. More likely, the signal triggering hydraulic adjustment either does not originate in the stomata or is impaired by the secondary changes caused by hypoxia.
It appears that the number of ARs, not its overall length and dry mass, was the overriding factor in maintaining root K r in hypoxic tobacco plants. Even though root porosity was similar in aerated and hypoxia treatments, the number of ARs was increased by hypoxia. The formation of ARs is an important adaptation to low O 2 conditions in some plants [2,34]. In the present study, hypoxia induced the formation of over twice as many ARs compared with aerated tobacco plants, but their length was reduced by hypoxia. Similar results were previously reported in tobacco and the authors concluded that the formation of short ARs cannot functionally replace the primary root system which contributed to the relative intolerance of tobacco to O 2 deficiency [37]. However, in the present study, K r in hypoxic plants showed no change compared with aerated plants. Additionally, P n and E of hypoxic plants showed no further decrease after one week of hypoxia compared with two days of treatment. These results indicate that the ARs were effective in maintaining root water transport. The roles of hypoxiainduced ARs are not limited to replacing existing roots. ARs induced by hypoxia had higher porosity than the LRs and could conduct more air from shoots to roots and the rhizosphere (radial O 2 loss, ROL). ROL of ARs may have a profound influence on rhizosphere aeration and nutrient availability in plants under O 2 deficiency [60]. Thus, the numerous short ARs induced by hypoxia in this study could potentially affect tobacco rhizosphere aeration and contribute to the survival and functioning of the root system under hypoxia.
Decreased root hydraulic conductivity of anoxic Arabidopsis has been linked to aquaporin closure [23], and a structure-based protonation mechanism under O 2 deprivation conditions has been demonstrated [23,24]. In addition to aquaporin gating analysis, aquaporin gene expression patterns can reveal the importance of various aquaporins in plant responses to O 2 deficiency [19]. Although transcripts may not be translated under certain conditions and posttranslational regulation can modify the function of aquaporins, transcriptional responses can shed light on the translational potential of cells experiencing O 2 deficiency [61]. Microarray analysis showed that aquaporin expression was downregulated in O 2 deficient Arabidopsis [29] and Persea americana [62]. Here, the relative transcript abundance of PIPs in ARs was compared with LRs. After two days of hypoxia, relative transcript abundance of PIP1;1 and PIP1;3 decreased in LRs but showed no change in ARs. One week of hypoxia resulted in decreased relative transcript abundance of PIPs in LRs except PIP1;1 and PIP1;3, while hypoxic ARs showed unchanged expression of PIPs on day seven. These results indicate that the relative transcript abundance of PIPs in ARs was less affected by hypoxia, which may contribute to hydraulic adjustment. In addition to regulating water transport, PIPs impact other physiological processes. Tobacco PIP1;2 has been reported to be permeable to CO 2 [25]. CO 2 can accumulate in roots of plants growing in stagnant water due to its low diffusion rate in water and high concentrations of CO 2 can cause cell acidification [60]. Thus, the down-regulation of PIP1;2 may lead to decreased efflux of CO 2 in cells and intensify cell acidification. Interestingly, expression of PIP1;1 and PIP1;3 in LRs showed different responsive patterns compared with the other examined PIP genes under hypoxia. Further research is needed to shed more light on the biological roles of PIP1;1 and PIP1;3 in tobacco. Tobacco PIP1;3 has been shown to be potentially involved in the O 2 transmembrane transport [28]. Although the experimental set-up of the two experiments are similar, the objective of this study was mainly comparing the differences between ARs and LRs under hypoxic conditions. However, unlike the present study, it was reported that the relative transcript abundance of tobacco PIP1;3 was upregulated by root hypoxia [28]. The reason for the difference may be that in the present study LRs and newly formed ARs were sampled separately rather than the whole root. Additionally, gene expression patterns in response to abiotic stresses may vary between different developmental stages [63]. Tobacco plants were exposed to root hypoxia three weeks after germination in the present study while plants were subjected to root hypoxia two weeks after germination in the study of Zwiazek et al. [28], which may also contribute to the differences in results. Since ARs were less affected by hypoxia than LRs in terms of PIP transcription, ARs can likely more actively participate in root water uptake under hypoxic conditions. In fact, ARs induced by hypoxia in Larix laricina were shown to have higher hydraulic conductivity than the existing roots [44]. However, it should be emphasized that both transcriptional and post-transcriptional regulation may affect aquaporin abundance and functions and several studies have shown the lack of correlation between aquaporin mRNA and protein abundance [64][65][66]. It remains to be determined whether the changes in transcript abundance observed in the present study are functionally significant.
Phytohormones, especially ethylene, are involved in the regulation network in response to O 2 deficiency, including the initiation and regulation of ARs [67,68]. In this study, transcript profiling showed that more ACS transcripts were induced in hypoxic LRs than ARs, which could result in the accumulation of ACC in hypoxic LRs. ACC, an ethylene synthesis precursor, is induced in flooded roots while the conversion of ACC to ethylene needs the presence of O 2 [69]. The transport of ACC from stressed roots to other tissues serves as a signal and causes the formation of ARs in aerated shoots [51]. Both ADH and PDC were frequently reported to be up-regulated in O 2 deficient tissues [13,62]. In this study, transcripts of tobacco ADH1 and PDC1 were sharply up-regulated in hypoxic LRs, while ARs showed much lower expression. Despite the inefficiency of ethanolic fermentation, the activities of ADH and PDC are essential to plants under low O 2 conditions to meet the energy demand [3]. However, the accumulation of end products of fermentation is toxic [3]. In the present study, two days of hypoxia resulted in a significant decline of ATP concentrations in both ARs and LRs. Although hypoxic ARs did not differ from hypoxic LRs in ATP concentration, hypoxic ARs maintained markedly lower levels of ADH and PDC transcripts compared with LRs. This suggests that the better aeration of ARs reduced their dependence on inefficient and toxic fermentation compared with LRs under low O 2 conditions.
In conclusion, the formation of short ARs was induced by hypoxia in hydroponically grown tobacco plants. Although, the length of ARs was reduced in hypoxic plants, it appears that they were likely more effective in facilitating O 2 diffusion into the roots and maintaining low level of fermentation and high K r . PIP expression patterns likely reflect the metabolic status in roots and may also be related to gas transport. Stable PIP transcripts of ARs under hypoxic conditions may be indicative of an active role of ARs in root water transport under hypoxic conditions. Supporting information S1 Dataset. Parameters recorded in this study. (XLSX) S1 Table. qRT-PCR primers for tobacco (Nicotiana tabacum) genes.