The novel and taxonomically restricted Ah24 gene from grain amaranth (Amaranthus hypochondriacus) has a dual role in development and defense

Abstract

Grain amaranths tolerate stress and produce highly nutritious seeds. We have identified several (a)biotic stress-responsive genes of unknown function in Amaranthus hypochondriacus, including the so-called Ah24 gene. Ah24 was expressed in young or developing tissues; it was also strongly induced by mechanical damage, insect herbivory and methyl jasmonate and in meristems and newly emerging leaves of severely defoliated plants. Interestingly, an in silico analysis of its 1304 bp promoter region showed a predominance of regulatory boxes involved in development, but not in defense. The Ah24 cDNA encodes a predicted cytosolic protein of 164 amino acids, the localization of which was confirmed by confocal microscopy. Additional in silico analysis identified several other Ah24 homologs, present almost exclusively in plants belonging to the Caryophyllales. The possible function of this gene in planta was examined in transgenic Ah24 overexpressing Arabidopsis thaliana and Nicotiana tabacum plants. Transformed Arabidopsis showed enhanced vegetative growth and increased leaf number with no penalty in one fitness component, such as seed yield, in experimental conditions. Transgenic tobacco plants, which grew and reproduced normally, had increased insect herbivory resistance. Modified vegetative growth in transgenic Arabidopsis coincided with significant changes in the expression of genes controlling phytohormone synthesis or signaling, whereas increased resistance to insect herbivory in transgenic tobacco coincided with higher jasmonic acid and proteinase inhibitor activity levels, plus the accumulation of nicotine and several other putative defense-related metabolites. It is proposed that the primary role of the Ah24 gene in A. hypochondriacus is to contribute to a rapid recovery post-wounding or defoliation, although its participation in defense against insect herbivory is also plausible.

Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms

“…Cultivation procedures for the germination, growth and maintenance of wild type (WT) and transgenic OE A . thaliana plants under controlled growing conditions were performed as described previously [ 14 , 15 ].…”

“…hypochondriacus plants were subjected to salt and water-deficit stress to confirm the induced expression of the AhERF and AhDof genes observed in silico . The experiments were performed in a growth chamber under controlled conditions of light (photosynthetic photon flux density ≈ 300 μmol m -2 s -1 ) and temperature (28°C, 16 h light/ 8 h dark), as described previously [ 14 , 15 , 33 ]. Leaf and root samples were collected at different after being exposed for different time periods (i. e, 2 to 8 days) to the stress conditions.…”

“…Primer design for these genes was performed using the DNA calculator software (Sigma-Aldrich St. Louis, MO, USA) and included part of the unique 3’ non-coding segments of the AhDof cDNA, and segments that did not overlap with genes sharing similarity with AhERF , to ensure specificity. The protocol followed for all qRT-PCR analysis is described elsewhere [ 3 , 14 , 33 ].…”

“…The ongoing characterization of a selected number of these genes has already identified genes with interesting characteristics associated with both growth and stress tolerance, such as the Ah24 orphan gene and the AhNF-YC transcription factor (TF) gene. Both genes were shown to significantly influence growth and increase tolerance to both biotic and abiotic stresses when overexpressed in tobacco and Arabidopsis [ 14 , 15 ].…”

Varying water deficit stress (WDS) tolerance in grain amaranths involves multifactorial shifts in WDS-related responses

“…It was previously shown that the WDS response in Ahypo roots included the accumulation of osmolytes and increased levels of ROS scavenging and heat shock proteins, together with the induction of certain TFs (Huerta-Ocampo et al , 2011). Several other amaranth genes have been subsequently proposed as possible contributing factors to increased tolerance against several (a)abiotic stresses in grain amaranth, including an orphan gene (Massange-Sánchez et al , 2015), a gene with an unknown function domain (Palmeros-Súarez et al , 2017) and various TF genes (Palmeros Súarez et al , 2015; Massange-Sánchez et al , 2016). The above genes were induced in grain amaranth by several stress conditions and frequently conferred stress tolerance when overexpressed in Arabidopsis plants.…”