Genome-Wide Characterization of AspATs in Populus: Gene Expression Variation and Enzyme Activities in Response to Nitrogen Perturbations

Abstract

Aspartate aminotransferase (AspAT) catalyzes a reversible transamination reaction between glutamate and oxaloacetate to yield aspartate and 2-oxoglutarate, exerting a primary role in amino acid biosynthesis and homeostasis of nitrogen (N) and carbon metabolism within all cellular organisms. While progress in biochemical characterization of AspAT has been made for decades, the molecular and physiological characteristics of different members of the AspAT gene family remain poorly known particularly in forest trees. Here, extensive genome-wide survey of AspAT encoding genes was implemented in black cottonwood (Populus trichocarpa Torr. & A. Gray), a model species of woody plants. Thorough inspection of the phylogenies, gene structures, chromosomal distribution, cis-elements, conserved motifs, and subcellular targeting resulted in the identification of 10 AspAT isogenes (PtAspAT1-10) in the Populus genome. RNA-seq along with quantitative real-time polymerase chain reaction (qRT-PCR) validation revealed that PtAspATs displayed diverse patterns of tissue-specific expression. Spatiotemporal expressions of homologous AspATs in the poplar hybrid clone ‘Nanlin895’ were further evaluated, showing that gene expressions varied depending on source-sink dynamics. The impact on AspAT transcripts upon N starvation and seasonal senescence showed the upregulation of five AspAT in leaves concurrent with drastic downregulation of six or more AspATs in roots. Additionally, marked reductions of many more AspATs transcripts were observed in roots upon N excess. Accordingly, AspAT activities were significantly suppressed upon N starvation by an in-gel assay, prompting the argument that enzyme activity was a more direct indicator of the growth morphology under a N stress regime. Taken together, the expression profiling and enzyme activities upon stress cues provide a theoretical basis for unraveling the physiological significance of specific gene(s) in regulation of N acquisition and remobilization in woody plants.

Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

“…Each reaction was performed in triplicate, and the amplification products were examined by agarose gel electrophoresis and melting curve analysis. The expression stability of reference genes ( actin and ubiquitin ) were assessed using GeNorm algorithms, and the relative gene expression level was calculated by normalizing the geometric mean of the reference genes, according to a previously described method [33,34]. Primers for reference genes and target genes are presented in Table S1.…”

Morphological and physiological plasticity response to low nitrogen stress in black cottonwood (Populus deltoides Marsh.)

“…This suggests that, with the change of N levels in the environment, the internal resource allocation by the plant changed adaptively (Luo et al 2019). In addition, chlorophyll concentrations, leaf area, net photosynthetic rates and enzyme activities have been shown to be less under low N conditions, which eventually leads to reductions in biomass (Li et al 2012;Luo et al 2015Luo et al , 2019Lu et al 2019;Su et al 2019).…”

Functional Characterization of Invertase Inhibitors PtC/VIF1 and 2 Revealed Their Involvements in the Defense Response to Fungal Pathogen in Populus trichocarpa

“…Plant Materials, Growth Conditions, and Stress Treatments P. trichocarpa (genotype Nisqually-1) grows on standard pot in the growth chamber, using a temperature cycling between 22°C (night) and 26°C (day) under long-day conditions (16 h light/8 h dark, 20 mE) according to a previous report (Li et al, 2017;Su et al, 2019). N. benthamiana and A. thaliana (ecotype Col-0) plants were maintained in a growth chamber at 25°C under a light regime of 16 h and 200-300 mE of long-day conditions.…”

“…After a culturing for five weeks, seedlings were transferred to standard pots with mixtures of vermiculite:perlite:peat (1:1:3). For the infection of the fungal pathogen, roots peripheral areas of 8-week-cultured plants were inoculated with 20 ml F. solani spore suspensions (2.0 × 10 6 spore/ml) for 48 and 72 h. Similarly, plants were irrigated with 20 ml ABA (100 mM, dissolved in 10% ethanol) once a day for 4 days, and grown for 48 and 96 h. The drought stress was induced by water withholding treatments for 96 and 120 h. For wounding treatments, the mature leaves were physically punched and harvested after 2 and 6 h. Seasonal senescence leaves were harvested from plants grown in a growth chamber according to a previous study (Su et al, 2019). The frozen samples were ground in the liquid nitrogen and subjected to RNA and protein extraction, followed by qRT-PCR and functional assay.…”

“…Duplicate or triplicate samples of P. trichocarpa were used for microarray analysis (Wilkins et al, 2009). For the qRT-PCR analyses, RNA extraction and cDNA synthesis were performed according to the previous report (Han et al, 2013;Su et al, 2019). Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, China).…”