Molecular and phenotypic characterization of transgenic wheat and sorghum events expressing the barley alanine aminotransferase

Abstract

Main conclusion -The expression of a barley alanine aminotransferase gene impacts agronomic outcomes in a C3 crop, wheat.The use of nitrogen-based fertilizers has become one of the major agronomic inputs in crop production systems. Strategies to enhance nitrogen assimilation and flux in planta are being pursued through the introduction of novel genetic alleles. Here an Agrobacterium-mediated approach was employed to introduce the alanine aminotransferase from barley (Hordeum vulgare), HvAlaAT, into wheat (Triticum aestivum) and sorghum (Sorghum bicolor), regulated by either constitutive or root preferred promoter elements. Plants harboring the transgenic HvAlaAT alleles displayed increased alanine aminotransferase (alt) activity. The enhanced alt activity impacted height, tillering and significantly boosted vegetative biomass relative to controls in wheat evaluated under hydroponic conditions, where the phenotypic outcome across these parameters varied relative to time of year study was conducted. Constitutive expression of HvAlaAT translated to elevation in wheat grain yield under field conditions. In sorghum, expression of HvAlaAT enhanced enzymatic activity, but no changes in phenotypic outcomes were observed. Taken together these results suggest that positive agronomic outcomes can be achieved through enhanced alt activity in a C3 crop, wheat. However, the variability observed across experiments under greenhouse conditions implies the phenotypic outcomes imparted by the HvAlaAT allele in wheat may be impacted by environment.

Improving Nitrogen Use Efficiency Through Overexpression of Alanine Aminotransferase in Rice, Wheat, and Barley

“…In these latter works, metabolic profiling yielded inconclusive results, and transcriptome analysis did not indicate a change in N transport and assimilation. Transgenic sugarcane and wheat plants with OsAnt1:HvAlaAT also showed improved NUE and/or biomass production (Snyman et al, 2015;Peña et al, 2017). Our current study is an extension of the previous works in rice as mentioned above, including additional physiological analyses on N influx, and in-depth metabolomics and transcriptomic profiling.…”

An ancestral NB-LRR with duplicated 3′UTRs confers stripe rust resistance in wheat and barley

“…We transformed wheat and barley with the fosmid PC1104 ( = F2-1; Supplementary Table 4), which has a 40-kb genomic fragment with NLR 4DS-1 , RLK 4DS-1 and RLK 4DS-2 . The spring wheat CB037 34 was selected as the primary wheat recipient genotype because it is highly susceptible (IT scores = 7–9) to 11 Pst races, including PSTv-4, PSTv-14, PSTv-37, PSTv-39, PSTv-40, PSTv-47, PSTv-143, PSTv-221, PSTv-239, PSTv-306, and PSTv-353 (Supplementary Data 1). After bombarding 1,590 wheat immature embryos, we obtained 24 putative transgenic plants.…”

Targeting Nitrogen Metabolism and Transport Processes to Improve Plant Nitrogen Use Efficiency

“…As indicated by framed white, green, purple, or blue backgrounds, the transgenic strategies used either (1) tissue-specific gene manipulation, (2) concurrent expression of a particular gene in both root and shoot tissues (OsNRT1.1A, Wang et al, 2018b;OsNRT1.1B, Hu et al, 2015;OsNRT2.1, Chen et al, 2016), (3) co-expression of two genes in root and shoot tissues (OsAMT1;2 and OsGOGAT1, Lee et al, 2020a), or (4) constitutive gene expression throughout the plant. Positive results were achieved with (a) native expression of ammonium transporters (OsAMT1-1, Ranathunge et al, 2014;OsAMT1;2, Lee et al, 2020a), nitrate transporters (OsNRT1.1A, Wang et al, 2018b;OsNRT1.1B, Hu et al, 2015;OsNRT2.1, Chen et al, 2016;OsNRT2.3b, Fan et al, 2016b;OsNPF7.3, Fang et al, 2017;OsNPF8.20, Fang et al, 2013), and amino acid transporters (OsAAP1, Ji et al, 2020;OsAAP6, Peng et al, 2014;GmAAP6a, Liu et al, 2020) in rice (Os), Arabidopsis (At), and soybean (Gm), (b) expression of a barley alanine aminotransferase (HvAlaAT) in canola (Good et al, 2007), rice (Shrawat et al, 2008;Beatty et al, 2013), sugarcane (Snyman et al, 2015), and wheat (Peña et al, 2017), and (c) by knocking out native/endogenous genes (Ataap2, Perchlik and Tegeder, 2018;Osaap3, Lu et al, 2018;Osaap5, Wang et al, 2019). Plants were grown under high (HN), moderate (MN), sufficient (SN; N supply was not specified) and low N (LN), or without N (-N).…”

“…On the other hand, expression of Escherichia coli EcasnA in Brassica napus (canola) seems to only be successful at high N status (Seiffert et al, 2004). AlaAT is involved in alanine synthesis and catabolism, and the constitutive or root-specific expression of barley HvAlaAT resulted in improved seed yields in wheat (Peña et al, 2017), or in canola (Good et al, 2007) and rice (Shrawat et al, 2008) (Figure 1). In addition, NUE was increased in rice, canola, and Saccharum officinarum (sugarcane) when HvAlaAT expression was targeted to roots and engineered plants were exposed to high N (Beatty et al, 2013) and/or low N (Good et al, 2007;Snyman et al, 2015).…”