Functional characterization of GPC-1 genes in hexaploid wheat

Abstract

In wheat, monocarpic senescence is a tightly regulated process during which nitrogen (N) and micronutrients stored pre-anthesis are remobilized from vegetative tissues to the developing grains. Recently, a close connection between senescence and remobilization was shown through the map-based cloning of the GPC (grain protein content) gene in wheat. GPC-B1 encodes a NAC transcription factor associated with earlier senescence and increased grain protein, iron and zinc content, and is deleted or non-functional in most commercial wheat varieties. In the current research, we identified 'loss of function' ethyl methanesulfonate mutants for the two GPC-B1 homoeologous genes; GPC-A1 and GPC-D1, in a hexaploid wheat mutant population. The single gpc-a1 and gpc-d1 mutants, the double gpc-1 mutant and control lines were grown under field conditions at four locations and were characterized for senescence, GPC, micronutrients and yield parameters. Our results show a significant delay in senescence in both the gpc-a1 and gpc-d1 single mutants and an even stronger effect in the gpc-1 double mutant in all the environments tested in this study. The accumulation of total N in the developing grains showed a similar increase in the control and gpc-1 plants until 25 days after anthesis (DAA) but at 41 and 60 DAA the control plants had higher grain N content than the gpc-1 mutants. At maturity, GPC in all mutants was significantly lower than in control plants while grain weight was unaffected. These results demonstrate that the GPC-A1 and GPC-D1 genes have a redundant function and play a major role in the regulation of monocarpic senescence and nutrient remobilization in wheat.

Fig. 1

Differences in senescence between GPC-1 mutants and control plants. (a) Flag leaf chlorophyll content in the TAU 2012 field experiment; Control (Δ), gpc-d1 mutant (◆), gpc-a1 mutant (▲), gpc-1 double mutant (●). The experiment consisted of five completely randomized blocks, for each genotype in each block 10 plants were tagged at anthesis and measured. Data is mean value (n=5 ± SE). (b) Difference in senescence at TAU 2012 between control plants (left) and gpc-1 double mutant plants (right) at 53 days after anthesis (DAA)

Fig. 2

Grain protein content (g kg⁻¹) of GPC-1 mutant and control plants grown in 2011 (left) and 2012 (right) winter growing season in three locations in Israel (TAU, KM and NY) and in one location in the USA (UCD), data is mean value (n=5 ± SE). Asterisks indicate significance compared to control using Tukey-HSD test for multiple comparisons. *P<0.05, ***P<0.001

Fig. 3

N accumulation in different tissues and time points. (a) total N accumulation (mg spike⁻¹) during grain development at 10, 25, 41 and 60 DAA, of gpc-1 (●) and control (Δ) collected at NY 2012 experiment. N levels (%) in gpc-1 and control plants was measured in (b) flag leaves at 50 DAA from a pot experiment at TAU 2013 and (c) peduncles collected at harvest time (completely yellow) at NY 2012 experiment. Data is mean value ± SE and asterisks indicate significance using a t-test. **P<0.01, ***P<0.001

Fig. 4

Validation of putative GPC-regulated genes. The relative expression levels of eight isogroups taken from Cantu et al. (2011) were compared in control (Δ) and gpc-1 double mutants (●) across a senescing time course by qRT-PCR. Y-axis values represent the fold-ACTIN level of expression of each isogroup ± SE based on ten biological replicates. Asterisks indicate significance using a t-test. * P<0.05, ** P<0.01, *** P<0.001. H = Heading date, D = Days after anthesis