An amaranth DGR gene, induced under abiotic stress, modifies cell wall structure and causes hypersensitivity to ABA and salt when overexpressed in Arabidopsis. DUF642 is a highly conserved plant-specific family of unknown cell wall-associated proteins. The AhDGR2 gene, coding for a DUF642 protein, was significantly induced in grain amaranth (Amaranthus hypochondriacus) plants subjected to water-deficit and salinity stress, thereby suggesting its participation in abiotic stress tolerance in this plant. A role in development was also inferred from the higher AhDGR2 expression rates detected in young tissues. Subsequent overexpression of AhDGR2 in transgenic Arabidopsis plants (OE-AhDGR2) supported its possible role in development processes. Thus, OE-AhDGR2 plants generated significantly longer roots when grown in normal MS medium. However, they showed a hypersensitivity to increasing concentrations of abscisic acid or NaCl in the medium, as manifested by shorter root length, smaller and slightly chlorotic rosettes, as well as highly reduced germination rates. Contrary to expectations, OE-AhDGR2 plants were intolerant to abiotic stress. Moreover, cell walls in transgenic plants were thinner, in leaves, and more disorganized, in roots, and had significantly modified pectin levels. Lower pectin methylesterase activity detected in leaves of OE-AhDGR2 plants, but not in roots, was contrary to previous reports associating DUF642 proteins and decreased pectin esterification levels in cell walls. Nonetheless, microarray data identified candidate genes whose expression levels explained the phenotypes observed in leaves of OE-AhDGR2 plants, including several involved in cell wall integrity and extension, growth and development, and resistance to abiotic stress. These results support the role of DUF642 proteins in cell wall-related processes and offer novel insights into their possible role(s) in plants.
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.
Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms.
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