Dynamics of nonstructural carbohydrates in a deciduous woody species from tropical dry forests under recurrent water deficit.

In tropical dry forests, both the dry and the short rainy seasons have become increasingly irregular. This study replicated these conditions to investigate the effects of two water deficit cycles on Cenostigma microphyllum seedlings. Impacts were assessed by measuring growth traits, water relations, gas exchange, and dynamics of nonstructural carbohydrate (NSC) content in the whole plant under greenhouse conditions in potted plants. In the first water deficit cycle, the leaf relative water content (RWC) was maintained at the expense of a rapid drop in gas exchange. Furthermore, there was a slight accumulation of NSC, mainly soluble sugars (SS) in the stem wood and roots, to the detriment of height and stem diameter growth. In the second cycle, the leaf RWC remained 40% higher than the lowest level measured in the first water deficit, and CO2 assimilation remained twice as long in previously stressed plants. The SS content of the stems and roots was strongly correlated with the predawn leaf RWC. No strong reduction was observed in the bark stock even with the gradual increase of SS in the wood. Our data suggest that under recurrent water deficit prior to leaf drop, CO2 assimilation is maintained, with the highest possible leaf RWC, under reduced stomatal conductance. This assists in SS transport to wood and root, which is no longer used to support the growth of the aboveground parts.

Stomatal Responses to Light, CO2, and Mesophyll Tissue in Vicia faba and Kalanchoë fedtschenkoi.

The responses of stomatal aperture to light intensity and CO2 concentration were studied in both Vicia faba (C3) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C3 in response to light and CO2. By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C3. This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C3 and CAM species.

Salt tolerance of Calotropis procera begins with immediate regulation of aquaporin activity in the root system.

The ability to respond quickly to salt stress can determine the tolerance level of a species. Here, we test how rapidly the roots of Calotropis procera react to high salinity conditions. In the first 24 h after saline exposure, the plants reduced stomatal conductance, increased CO2 assimilation, and water use efficiency. Thus, the root tissue showed an immediate increase in soluble sugars, free amino acid, and soluble protein contents. Twelve aquaporins showed differential gene expression in the roots of C. procera under salinity. Transcriptional upregulation was observed only after 2 h, with greater induction of CpTIP1.4 (fourfold). Transcriptional downregulation, in turn, occurred mainly after 8 h, with the largest associated with CpPIP1.2 (fourfold). C. procera plants responded quickly to high saline levels. Our results showed a strong stomatal control associated with high free amino acid and soluble sugar contents, regulated aquaporin expression in roots, and supported the high performance of the root system of C. procera under salinity. Moreover, this species was able to maintain a lower Na+/K+ ratio in the leaves compared to that of the roots of stressed plants. The first response of the root system, after immediate contact with saline solution, present an interesting scenario to discuss. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00957-9.

Reference genes selection for Calotropis procera under different salt stress conditions.

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

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.

Seasonal variability in physiological and anatomical traits contributes to invasion success of Prosopis juliflora in tropical dry forest.

We investigated whether there were consistent differences in the physiological and anatomical traits and phenotypic variability of an invasive (Prosopis juliflora (Sw.) DC.) and native species (Anadenanthera colubrina (Vell.) Brenan) in response to seasonality in a tropical dry forest. The water potential, organic solutes, gas exchange, enzymes of the antioxidant system, products of oxidative stress and anatomical parameters were evaluated in both species in response to seasonality. An analysis of physiological responses indicated that the invasive P. juliflora exhibited higher response in net photosynthetic rate to that of the native species between seasons. Higher values of water potential of the invasive species than those of the native species in the dry season indicate a more efficient mechanism for water regulation in the invasive species. The invasive species exhibits a thicker cuticle and trichomes, which can reduce transpiration. In combination, the increased epidermal thickness and the decreased thickness of the parenchyma in the dry season may contribute to water saving. Our data suggest a higher variability in anatomical traits in the invasive species as a response to seasonality, whereas physiological traits did not present a clear pattern of response.

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.

Overcoming seed dormancy using gibberellic acid and the performance of young Syagrus coronata plants under severe drought stress and recovery.

Syagrus coronata, a native palm tree of the Brazilian semi-arid region, exhibits low germinability due to seed dormancy. This study aimed to increase the germinability, analyze the morphology of seedlings and evaluate the performance of young plants under a water deficit. We used immersion in water and gibberellic acid (GA3) as pyrene (seed with endocarp) pre-germination treatments, and we analyzed the water relations, gas exchange, chlorophyll fluorescence and carbon balance components of young plants under drought and rehydration conditions. The immersion of pyrenes in 0.3 mM GA3 solution for 24 h enhanced the emergence and survival of plants and the emergence rate index. The germination of S. coronata is of the remote tubular type, and seedling growth originates with the protrusion of the cotyledon petiole, followed by the subsequent emergence of the root, leaf sheaths and eophyll. The plants exhibited high tolerance to no irrigation for 37 days, which was attributed to strong stomatal control, a higher proportion of energy dissipation and a higher content of photoprotective pigments. Despite the reduced stomatal conductance (regardless of soil water availability), the photosynthetic rate remained high throughout the day, which indicated a low correlation between these two parameters. After rehydration, we observed that both the leaf water content and photosynthesis recovered, which showed an absence of irreversible damage of the photosynthetic apparatus. The use of 0.3 mM GA3 is recommended as a treatment for overcoming seed dormancy in this species. Young S. coronata plants showed high tolerance during drought and resilience after rehydration by adjusting their leaf metabolism, which could explain the endemism of this species in semi-arid regions and its ability to remain evergreen throughout the year. Furthermore, with high photosynthetic rate in the most favorable time of day, even under drought stress.

Three cycles of water deficit from seed to young plants of Moringa oleifera woody species improves stress tolerance.

The main objective of this study was to assess whether recurring water stress occurring from seed germination to young plants of Moringa oleifera Lam. are able to mitigate the drought stress effects. Germination, gas exchange and biochemical parameters were analysed after three cycles of water deficit. Young plants were used 50 days after germination under three osmotic potentials (0.0, -0.3 and -0.4 MPa). For each germination treatment, control (irrigated) and stressed (10% of water control) plants were compared for a total of six treatments. There were two cycles of drought interspersed with 10 days of rehydration. The young plants of M. oleifera showed increased tolerance to repeated cycles of drought, maintaining high relative water content (RWC), high water use efficiency (WUE), increased photosynthetic pigments and increased activity of antioxidant enzymes. There was rapid recovery of the photosynthetic rate during the rehydration period. The stressed plants from the -0.3 and -0.4 MPa treatments showed higher tolerance compared to the control plants. The results suggest that seeds of M. oleifera subjected to mild water deficit have had increased the ability for drought tolerance when young plant.

Seasonal effects on the relationship between photosynthesis and leaf carbohydrates in orange trees.

To understand the effect of summer and winter on the relationships between leaf carbohydrate and photosynthesis in citrus trees growing in subtropical conditions, 'Valencia' orange trees were subjected to external manipulation of their carbohydrate concentration by exposing them to darkness and evaluating the maximal photosynthetic capacity. In addition, the relationships between carbohydrate and photosynthesis in the citrus leaves were studied under natural conditions. Exposing the leaves to dark conditions decreased the carbohydrate concentration and increased photosynthesis in both seasons, which is in accordance with the current model of carbohydrate regulation. Significant negative correlations were found between total non-structural carbohydrates and photosynthesis in both seasons. However, non-reducing sugars were the most important carbohydrate that apparently regulated photosynthesis on a typical summer day, whereas starch was important on a typical winter day. As a novelty, photosynthesis stimulation by carbohydrate consumption was approximately three times higher during the summer, i.e. the growing season. Under subtropical conditions, citrus leaves exhibited relatively high photosynthesis and high carbohydrate levels on the summer day, as well as a high nocturnal consumption of starch and soluble sugars. A positive association was determined between photosynthesis and photoassimilate consumption/exportation, even in leaves showing a high carbohydrate concentration. This paper provides evidence that photosynthesis in citrus leaves is regulated by an increase in sink demand rather than by the absolute carbohydrate concentration in leaves.

Approximate Entropy as a measure of complexity in sap flow temporal dynamics of two tropical tree species under water deficit.

Approximate Entropy (ApEn), a model-independent statistics to quantify serial irregularities, was used to evaluate changes in sap flow temporal dynamics of two tropical species of trees subjected to water deficit. Water deficit induced a decrease in sap flow of G. ulmifolia, whereas C. legalis held stable their sap flow levels. Slight increases in time series complexity were observed in both species under drought condition. This study showed that ApEn could be used as a helpful tool to assess slight changes in temporal dynamics of physiological data, and to uncover some patterns of plant physiological responses to environmental stimuli.