Dehydration response in Stylosanthes scabra: Transcriptional, biochemical, and physiological modulations.

Stylosanthes scabra, popularly known as stylo, is native to the Brazilian Caatinga semiarid region and stands out as a drought-tolerant shrub forage crop. This work provides information about the plant response during the first 48 h of water deficit, followed by a rehydration treatment. Besides root transcriptomics data, 13 physiological or biochemical parameters were scrutinized. Additionally, RNA-Seq annotated transcripts not associated with the "Viridiplantae" clade were taxonomically categorized. It was found that S. scabra quickly perceives and recovers from the oscillations of the imposed water regime. Physiologically, mechanisms that minimize evapotranspiration or protect the photosynthetic apparatus stood out. Biochemically, it was found that the root tissue invests in synthesizing compounds that can act as osmolytes (proline and sugars), emphasizing the importance of osmoregulation to water deficit acclimation. Consistently, transcriptome and qPCR analyses showed that a set of enriched biological processes with upregulated (UR) transcripts were involved in protective functions against reactive oxygen species or encoding enzymes of important metabolic pathways, which might contribute to S. scabra response to water deficit. Additionally, several UR kinases and transcription factors were identified. Finally, in an innovative approach, some naturally occurring microbial groups (such as Schizosaccharomyces, Bradyrhizobium, etc.) were identified in the S. scabra roots. This study reveals insights into the physiological, biochemical, and molecular mechanisms underlying the S. scabra response to water deficit and provides candidate genes that may be useful in developing drought-tolerant crop varieties through biotechnological applications.

Reference genes for quantitative real-time PCR normalization of Cenostigma pyramidale roots under salt stress and mycorrhizal association.

Cenostigma pyramidale is a native legume of the Brazilian semiarid region which performs symbiotic association with arbuscular mycorrhizal fungi (AMF), being an excellent model for studying genes associated with tolerance against abiotic and biotic stresses. In RT-qPCR approach, the use of reference genes is mandatory to avoid incorrect interpretation of the relative expression. This study evaluated the stability of ten candidate reference genes (CRGs) from C. pyramidale root tissues under salt stress (three collection times) and associated with AMF (three different times of salinity). The de novo transcriptome was obtained via RNA-Seq sequencing. Three algorithms were used to calculate the stability of CRGs under different conditions: (i) global (Salt, Salt+AMF, AMF and Control, and collection times), (ii) only non-inoculated plants, and (iii) AMF (only inoculated plants). HAG2, SAC1, aRP3 were the most stable CRGs for global and AMF assays, whereas HAG2, SAC1, RHS1 were the best for salt stress assay. This CRGs were used to validate the relative expression of two up-regulated transcripts in Salt2h (RAP2-3 and PIN8). Our study provides the first set of reference genes for C. pyramidale under salinity and AMF, supporting future researches on gene expression with this species.

Transcriptome of Cenostigma pyramidale roots, a woody legume, under different salt stress times.

Salinity stress has a significant impact on the gain of plant biomass. Our study provides the first root transcriptome of Cenostigma pyramidale, a tolerant woody legume from a tropical dry forest, under three different salt stress times (30 min, 2 h, and 11 days). The transcriptome was assembled using the RNA sequencing (RNA-Seq) de novo pipeline from GenPipes. We observed 932, 804, and 3157 upregulated differentially expressed genes (DEGs) and 164, 273, and 1332 downregulated DEGs for salt over 30 min, 2 h, and 11 days, respectively. For DEGs annotated with the Viridiplantae clade in the early stress periods, the response to salt stress was mainly achieved by stabilizing homeostasis of such ions like Na+ and K+ , signaling by Ca2+ , transcription factor modulation, water transport, and oxidative stress. For salt stress at 11 days, we observed a higher modulation of transcription factors including the WRKY, MYB, bHLH, NAC, HSF, and AP2-EREBP families, as well as DEGs involved in hormonal responses, water transport, sugar metabolism, proline, and reactive oxygen scavenging mechanisms. Five selected DEGs (K+ transporter, aquaporin, glutathione S-transferase, cyclic nucleotide-gated channel, and superoxide dismutase) were validated by qPCR. Our results indicated that C. pyramidale had an early perception of salt stress modulating ionic channels and transporters, and as the stress progressed, the focus turned to the antioxidant system, aquaporins, and complex hormone responses. The results of this first root transcriptome provide clues on how this native species modulate gene expression to achieve salt stress tolerance.

Remobilization of leaf Na+ content and use of nonstructural carbohydrates vary depending on the time when salt stress begins in woody species.

Basic mechanisms are known to promote salt tolerance in plants: a delay in Na+ uptake or rapid Na+ remobilization from leaf tissue. We measured dynamics of the Na+/K+ ratio and components of carbon metabolism during the first 72 h after saline stress (200 mM NaCl) began in Cenostigma pyramidale, a woody species, under controlled conditions. Saline stress at two times: one plant group at the beginning of the morning and the other in the evening. Stressed plants had three times more Na+ in leaves than did control plants in the first 24 h. However, in the next few hours, despite new applications of saline solution, the Na+/K+ ratio continued to decline. Several samples, including night treatments, provided evidence that this species uses Na+ recirculation mechanisms to endure salt stress. Effects of salt on the traits evaluated differed depending on the time when stress began. Between the two saline treatments, in the first 24 h after saline stress, gas exchange decreased more strongly in morning-stressed plants, when large amounts of Na+ reached the leaf and K+ left this organ. Nevertheless, when stress was applied in the evening, leaf Na+ remobilization was faster, and the soluble sugar/starch ratio remained greater than did the control. Our data suggested that time of the beginning of salt stress could change the level of damage. Morning-stressed plants synthesized greater amounts of proline, H2O2, and malondialdehyde than did night-stressed plants. We recommend that details regarding the time of stress be taken into consideration in physiological studies.

Corrigendum: Ecophysiological Traits of Invasive C3 Species Calotropis procera to Maintain High Photosynthetic Performance Under High VPD and Low Soil Water Balance in Semi-Arid and Seacoast Zones.

[This corrects the article DOI: 10.3389/fpls.2020.00717.].

Ecophysiological Traits of Invasive C3 Species Calotropis procera to Maintain High Photosynthetic Performance Under High VPD and Low Soil Water Balance in Semi-Arid and Seacoast Zones.

The evergreen C3 plant Calotropis procera is native to arid environments. Thus, it grows under high vapor pressure deficit (VPD), intense light, and severe drought conditions. We measured several ecophysiological traits in C. procera plants growing in semi-arid and seacoast environments to assess the attributes that support its photosynthetic performance under these contrasting conditions. Gas exchange analysis, primary metabolism content, nutrients, the antioxidant system, and leaf anatomy traits were measured under field conditions. In the semi-arid environment, C. procera was exposed to a prolonged drought season with a negative soil water balance during the 2 years of the study. Calotropis procera plants were exposed to a positive soil water balance only in the rainy season in the seacoast environment. The leaves of C. procera showed the same photosynthetic rate under high or low VPD, even in dry seasons with a negative soil water balance. Photosynthetic pigments, leaf sugar content, and the activity of antioxidant enzymes were increased in both places in the dry season. However, the anatomical adjustments were contrasting: while, in the semi-arid environment, mesophyll thickness increased in the driest year, in the seacoast environment, the cuticle thickness and trichome density were increased. The ability to maintain photosynthetic performance through the seasons would be supported by new leaves with different morpho-anatomical traits, with contrasting changes between semi-arid and seacoast environments. Furthermore, our results suggest that an efficient antioxidative system and leaf sugar dynamics can contribute to protecting the photosynthetic machinery even under severe drought.

Different resource-use strategies of invasive and native woody species from a seasonally dry tropical forest under drought stress and recovery.

Exotic plants in semiarid region have developed strategies for efficient use or capture of resources. They have become invasive and outperform native species. To understand which factors could explain the success of invasive woody species in a semiarid region, several physiological traits were analyzed in young plants of two invasive and two native species exposed to different water availability. Invasive plants showed low leaf construction cost, high phosphorus and nitrogen contents, reduced loss of instantaneous energy use efficiency, and smaller specific leaf area when compared to native species. This strategy led to a higher biomass gain and a high root/shoot ratio in both water treatments. After rehydration, invasive plants showed faster recovery and higher rates of CO2 assimilation. This resilience is fundamental for species in semiarid regions, and also increase uptake of nutrients. Maintaining a high photosynthetic rate, whenever there is water availability is a strategy that increases the performance of the species in relation to biomass gain. The low leaf construction cost and the fast recovery of the photosynthetic metabolism of invasive plants after limiting water resources explains the success of these species, and suggests that their potential may increase under prolonged and severe drought seasons.

Arbuscular mycorrhizal fungi improve photosynthetic energy use efficiency and decrease foliar construction cost under recurrent water deficit in woody evergreen species.

Plants suffer recurrent cycles of water deficit in semiarid regions and have several mechanisms to tolerate low water availability. Thus, arbuscular mycorrhizal fungi (AMF) can alleviate deleterious effects of stress. In this study, Cynophalla flexuosa plants, a woody evergreen species from semiarid, when associated with AMF were exposed to two consecutive cycles of water deficit. Leaf primary metabolism, specific leaf area (SLA), leaf construction cost (CC) and photosynthetic energy use efficiency (PEUE) were measured. The maximum stress occurred on seven days (cycle 1) and ten days (cycle 2) after suspending irrigation (photosynthesis close to zero). The rehydration was performed for three days after each maximum stress. In both cycles, plants submitted to water deficit showed reduced gas exchange and leaf relative water content. However, Drought + AMF plants had significantly larger leaf relative water content in cycle 2. At cycle 1, the SLA was larger in non-inoculated plants, while CC was higher in inoculated plants. At cycle 2, Drought + AMF treatment had lower CC and large SLA compared to control, and high PEUE compared to Drought plants. These responses suggest AMFs increase tolerance of C. flexuosa to recurrent water deficit, mainly in cycle 2, reducing the CC, promoting the improvement of SLA and PEUE, leading to higher photosynthetic area. Thus, our result emphasizes the importance of studies on recurrence of water deficit, a common condition in semiarid environments.

Arbuscular mycorrhizal fungi and foliar phosphorus inorganic supply alleviate salt stress effects in physiological attributes, but only arbuscular mycorrhizal fungi increase biomass in woody species of a semiarid environment.

Salinity may limit plant growth especially in arid and semiarid regions. Arbuscular mycorrhizal fungi (AMF) and the supply of inorganic phosphorus (Pi) could alleviate the negative effects of such stress by improvement in stomatal conductance, photosynthesis and biomass. The aim of this study is to evaluate the ecophysiological performance of Cenostigma pyramidale (Tul.) E. Gagnon & G. P. Lewis (Fabaceae) in a greenhouse under salinity conditions in combination with the supply of AMF and leaf Pi. The experiment was conducted in a factorial design considering two levels of salinity (+NaCl and -NaCl), two levels of AMF (+AMF and -AMF) and two levels of leaf Pi supply (+Pi and -Pi). The variables gas exchange, leaf primary metabolism, dry biomass and nutrients were measured. Plants with AMF under non-saline conditions presented a high photosynthesis and biomass. In saline conditions, AMF promoted lower decrease in photosynthesis, high shoot dry matter and low content of leaf and root Na+ and Cl-. Plants treated with leaf Pi increased biomass and photosynthetic pigments under both conditions and accumulated more Cl- in shoots under salinity conditions. When combined, AMF * Pi increased photosynthesis only in non-saline conditions. Plants under salinity conditions without AMF and Pi had higher decreases in gas exchange and high content of Cl- in roots. Therefore, C. pyramidale plants improved their metabolism under both growth conditions in the presence of AMF, Pi or a combination of both. However, the greatest increases in growth and tolerance to salinity occurred in the isolated presence of AMF.

Photosynthetic limitation and mechanisms of photoprotection under drought and recovery of Calotropis procera, an evergreen C3 from arid regions.

Calotropis procera is a C3 plant native from arid environmental zones. It is an evergreen, shrubby, non-woody plant with intense photosynthetic metabolism during the dry season. We measured photosynthetic parameters and leaf biochemical traits, such as gas exchange, photochemical parameters, A/Ci analysis, organic solutes, and antioxidant enzymes under controlled conditions in potted plants during drought stress, and following recovery conditions to obtain a better insight in the drought stress responses of C. procera. Indeed, different processes contribute to the drought stress resilience of C. procera and to the fast recovery after rehydration. The parameters analyzed showed that C. procera has a high efficiency for energy dissipation. The photosynthetic machinery is protected by a robust antioxidant system and photoprotective mechanisms such as alternative pathways for electrons (photorespiration and day respiration). Under severe drought stress, increased stomatal limitation and decreased biochemical limitation permitted C. procera to maintain maximum rate of Rubisco carboxylation (Vc,max) and photosynthetic rate (Amax). On the other hand, limitation of stomatal or mesophyll CO2 diffusion did not impair fast recovery, maintaining Vc,max, chloroplast CO2 concentration (Cc) and mesophyll conductance (gm) unchanged while electron flow used for RuBP carboxylation (Jc) and Amax increased. The ability to tolerate drought stress and the fast recovery of this evergreen C3 species was also due to leaf anti-oxidative stress enzyme activity, and photosynthetic pigments. Thus, these different drought tolerance mechanisms allowed high performance of photosynthetic metabolism by drought stressed plants during the re-watering period.

Symbiosis with AMF and leaf Pi supply increases water deficit tolerance of woody species from seasonal dry tropical forest.

In seasonal dry tropical forests, plants are subjected to severe water deficit, and the arbuscular mycorrhizal fungi (AMF) or inorganic phosphorus supply (Pi) can mitigate the effects of water deficit. This study aimed to assess the physiological performance of Poincianella pyramidalis subjected to water deficit in combination with arbuscular mycorrhizal fungi (AMF) and leaf inorganic phosphorus (Pi) supply. The experiment was conducted in a factorial arrangement of 2 water levels (+H2O and -H2O), 2 AMF levels (+AMF and -AMF) and 2Pi levels (+Pi and -Pi). Leaf primary metabolism, dry shoot biomass and leaf mineral nutrients were evaluated. Inoculated AMF plants under well-watered and drought conditions had higher photosynthesis and higher shoot biomass. Under drought, AMF, Pi or AMF+Pi plants showed metabolic improvements in photosynthesis, leaf biochemistry and higher biomass compared to the plants under water deficit without AMF or Pi. After rehydration, those plants submitted to drought with AMF, Pi or AMF+Pi showed a faster recovery of photosynthesis compared to treatment under water deficit without AMF or Pi. However, plants under the drought condition with AMF showed a higher net photosynthesis rate. These findings suggest that AMF, Pi or AMF+Pi increase the drought tolerance in P. pyramidalis, and AMF associations under well-watered conditions increase shoot biomass and, under drought, promoted faster recovery of photosynthesis.

Different mechanisms drive the performance of native and invasive woody species in response to leaf phosphorus supply during periods of drought stress and recovery.

The effects of drought stress and leaf phosphorus (Pi) supply on photosynthetic metabolism in woody tropical species are not known, and given the recent global environmental change models that forecast lower precipitation rates and periods of prolonged drought in tropical areas, this type of study is increasingly important. The effects of controlled drought stress and Pi supply on potted young plants of two woody species, Anadenanthera colubrina (native) and Prosopis juliflora (invasive), were determined by analyzing leaf photosynthetic metabolism, biochemical properties and water potential. In the maximum stress, both species showed higher leaf water potential (Ψl) in the treatment drought +Pi when compared with the respective control -Pi. The native species showed higher gas exchange under drought +Pi than under drought -Pi conditions, while the invasive species showed the same values between drought +Pi and -Pi. Drought affected the photochemical part of photosynthetic machinery more in the invasive species than in the native species. The invasive species showed higher leaf amino acid content and a lower leaf total protein content in both Pi treatments with drought. The two species showed different responses to the leaf Pi supply under water stress for several variables measured. In addition, the strong resilience of leaf gas exchange in the invasive species compared to the native species during the recovery period may be the result of higher efficiency of Pi use. The implications of this behavior for the success of this invasive species in semiarid environments are discussed.