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The evergreen C 3 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.

Ecophysiological traits of Prosopis juliflora (Sw.) DC. and a phylogenetically and ecologically similar native species, Anadenanthera colubrina (Vell.) Brenan, were studied to understand the invasive species’ success in caatinga, a seasonally dry tropical forest ecosystem of the Brazilian Northeast. To determine if the invader exhibited a superior resource-capture or a resource-conservative strategy, we measured biophysical and biochemical parameters in both species during dry and wet months over the course of two years. The results show that P. juliflora benefits from a flexible strategy in which it frequently outperforms the native species in resource capture traits under favorable conditions (e.g., photosynthesis), while also showing better stress tolerance (e.g., antioxidant activity) and water-use efficiency in unfavorable conditions. In addition, across both seasons the invasive has the advantage over the native with higher chlorophyll/carotenoids and chlorophyll a/b ratios, percent N, and leaf protein. We conclude that Prosopis juliflora utilizes light, water and nutrients more efficiently than Anadenanthera colubrina, and suffers lower intensity oxidative stress in environments with reduced water availability and high light radiation.

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) Leaf 100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root 50 and iii) Root 200 = 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 (Leaf 100 , Root 50, and Root 200 ). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes ( NAC78 , CNBL4 , and ND1 ) in Leaf 100 and Root 200 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.

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