Identification of predominant genes involved in regulation and execution of senescence-associated nitrogen remobilization in flag leaves of field grown barley

Abstract

The transcriptomes of senescing flag leaves collected from barley field plots with standard or high nitrogen supply were compared to identify genes specifically associated with nitrogen remobilization during leaf senescence under agronomically relevant conditions. In flag leaves collected in field plots with high nitrogen supply, the decline in chlorophyll content was delayed. By comparing changes in gene expression for the two nitrogen levels, it was possible to discriminate genes related to nitrogen remobilization during senescence and genes involved in other processes associated with the late development of leaves under field conditions. Predominant genes that were more strongly upregulated during senescence of flag leaves from plants with standard nitrogen supply included genes encoding the transcription factor HvNAC026, serine type protease SCPL51, and the autophagy factors APG7 and ATG18F. Elevated expression of these genes in senescing leaves from plants with standard nitrogen supply indicates important roles of the corresponding proteins in nitrogen remobilization. In comparison, the genes upregulated in both flag leaf samples might have roles in general senescence processes associated with late leaf development. Among these genes were the transcription factor genes HvNAC001, HvNAC005, HvNAC013, HvWRKY12 and MYB, genes encoding the papain-like cysteine peptidases HvPAP14 and HvPAP20, as well as a subtilase gene.

Keywords: Barley; Hordeum vulgare L.; HvNAC026.; field experiment; flag leaf; leaf senescence; nitrogen remobilization; nitrogen supply.

Fig. 3.

Relative expression levels of selected genes as analysed by quantitative real-time PCR. RNA samples were prepared from leaves of plants with standard nitrogen supply (SN) and high nitrogen supply (HN), respectively. Leaves were collected every fourth day from 12 June (12 days after flowering of SN and HN plants) until 4 July 2010. The relative chlorophyll content at 12 June was set to 100% (Supplementary Fig. S1A, available at JXB online). The expression data are scaled relative to the overall mean, error bars indicate SD and are derived from three biological replicates, n=3. (A) Controls and senescence marker, (B) genes showing higher expression in HN than in SN samples, and (C) genes showing higher expression in SN than in HN samples.

Fig. 1.

Characterization of flag leaves and ears collected from barley plants grown in field plots supplied with standard (SN) or high nitrogen (HN) in the period from 27 May (16 days after flowering of SN and HN plants) until 20 June 2009. Samples used for microarray analyses are highlighted in grey. (A) Relative chlorophyll content. Error bars indicate standard deviations (SD), n=80. (B) Protein content of leaves. Error bars indicate SD, n=8. (C) Dry weight of the ears. Error bars indicate SD, n=16.

Fig. 2.

(A) Microarray design used to analyse differences between non-senescing and senescing samples from plants grown under standard nitrogen (SN) or high nitrogen (HN) supply. Relative chlorophyll contents (Fig. 1A) are given in percentages. Each line represents one comparison. (B) Filtering of the microarray results. Conditions for each filtering step are given. The procedure resulted in two groups of genes: genes only differentially expressed in the comparison SN2 vs. SN1 and genes differentially expressed in both comparisons SN2 vs. SN1 and HN3 vs. HN1.