Histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 proteins affect plant growth under nitrogen deficient conditions in Arabidopsis thaliana

Abstract

Nitrogen (N) availability is one of the most important factors regulating plant metabolism and growth as it affects global gene expression profiles. Dynamic changes in chromatin structure, including histone modifications and nucleosome assembly/disassembly, have been extensively shown to regulate gene expression under various environmental stresses in plants. However, the involvement of chromatin related changes in plant nutrient responses has been demonstrated only in a few studies to date. In this study, we investigated the function of histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) proteins under N deficient conditions in Arabidopsis. In the nap1;1 nap1;2 nap1;3 triple mutant (m123-1), the expression of N-responsive marker genes and growth of lateral roots were decreased under N deficient conditions. In addition, the m123-1 plants showed a delay in N deficiency-induced leaf senescence. Taken together, these results suggest that NAP1s affect plant growth under N deficient conditions in Arabidopsis.

Nitrogen (N) availability is an essential factor for regulating plant metabolism, growth, and development (Crawford and Forde 2002; Gutiérrez 2012; Vidal et al. 2020). Plants mainly take up inorganic forms of N (nitrate and ammonium) from the soil and use them to synthesize indispensable organic compounds. In addition, nitrate is known to function as a signaling molecule, which reprograms global gene expression levels to optimize N uptake and metabolism (Bi et al. 2007; Konishi and Yanagisawa 2013; Liu et al. 2017; Marchive et al. 2013). Under N deficient conditions, plants upregulate the expression of genes encoding high affinity nitrate transporters and cytosolic glutamine synthetases, thus improving N acquisition efficiency and N utilization efficiency, respectively (Bernard and Habash 2009; Ishiyama et al. 2004; Lejay et al. 1999; Li et al. 2007; Masclaux-Daubresse et al. 2010; Zhuo et al. 1999).

Chromatin remodeling, including histone modifications and nucleosome assembly and disassembly, contributes to gene expression regulation (Klemm et al. 2019; Lai and Pugh 2017; Zaret and Mango 2016). Dynamic changes in chromatin structure have been observed in response to numerous environmental conditions (Asensi-Fabado et al. 2017; Chang et al. 2020; Chinnusamy and Zhu 2009; Lämke and Bäurle 2017). Post-translational modifications of histones not only affect chromatin structure but also lead to the recruitment of remodeling enzymes, which can regulate gene transcription (Bannister and Kouzarides 2011; Swygert and Peterson 2014). In mammals and plants, histone H3 can be mono-, di-, tri-methylated at specific lysine residues, which are associated with transcriptional activation (H3K4 and H3K36) or repression (H3K27 and H3K9) (Berr et al. 2011; Black et al. 2012; Liu et al. 2010; Martin and Zhang 2005; Xiao et al. 2016). Recently, several studies showed that histone modifications participate in nutrient acquisition in environmental conditions by regulating the changes in gene expression (Secco et al. 2017; Séré and Martin 2020). In Arabidopsis, expression of the high affinity nitrate transporter gene NITRATE TRANSPORTER 2.1 (NRT2.1) is upregulated at the onset of N deficiency (Filleur and Daniel-Vedele 1999; Lejay et al. 1999). A previous study identified HIGH NITROGEN-INSENSITIVE 9 (HNI9) as a key regulator of histone modification at the NRT2.1 locus (Widiez et al. 2011). HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex, which represses NRT2.1 expression in roots under high N conditions by modulating H3K27me3 level at the NRT2.1 locus (Widiez et al. 2011).

Nucleosome assembly and disassembly also play important roles in chromatin remodeling. The NUCLESOME ASSEMBLY PROTEIN 1 (NAP1) family of proteins act as primary chaperones of histones H2A and H2B, which are highly conserved among eukaryotes. NAP1 proteins bind to and shuttle histones into the nucleus, facilitating nucleosome assembly (Hammond et al. 2017; Park and Luger 2006; Zhou et al. 2015). The Arabidopsis genome harbors four genes encoding NAP1 proteins (NAP1;1 to NAP1;4) and two genes encoding NAP1-RELATED PROTEINS (NRPs) (NRP1 and NRP2) (Galichet and Gruissem 2006; Zhu et al. 2006). In Arabidopsis, NAP1 proteins play a redundant role in the response to different abiotic stresses, such as DNA damage and salt stress (Liu et al. 2009a, 2009b), and facilitate plant adaptation to phosphate starvation (Iglesias et al. 2013). The NAP1;1-NAP1;3 triple mutant plants exhibit lower expression levels of Pi starvation-induced genes, and consequently lower Pi accumulation, than wild-type Arabidopsis plants (Iglesias et al. 2013).

These studies indicate that chromatin remodeling plays an essential role in regulating plant responses to changes in environmental conditions including nutrient availability. However, the detailed mechanism of chromatin remodeling-mediated plant responses to N availability remains largely unknown. In this study, we investigated the functions of Arabidopsis NAP1 proteins under N deficient conditions.

To investigate the functions of NAP1s under N deficient conditions in Arabidopsis, we first examined the expression patterns of several N-responsive genes in triple loss-of-function mutant of NAP1;1-NAP1;3 m123-1 (Liu et al. 2009a) under different N contents. Seeds of Arabidopsis thaliana ecotype Columbia (Col-0) and m123-1 mutant were surface-sterilized and were grown on Murashige and Skoog (MS) medium supplemented with 100 mM glucose and different N concentrations under long-day (16 h light/8 h dark) photoperiod after dark incubation for two days at 4°C. Total RNA was isolated from 8-day-old whole wild-type (WT; Col-0) and m123-1 mutant seedlings grown on control N (30 mM N; 10 mM NH₄NO₃ and 10 mM KNO₃) and low N (0.3 mM N; 0.1 mM NH₄NO₃ and 0.1 mM KNO₃) media for 24 h before harvesting. Then, cDNA was synthesized as described previously (Sanagi et al. 2021), and quantitative real-time PCR (qRT-PCR) was performed on the Mx3000P QPCR System (Agilent Technologies) using gene-specific primers (Supplementary Table S1) and TB Green Premix Ex Taq II (TaKaRa), according to the manufacturer’s instructions. Expression levels of NRT2.1 and the cytosolic glutamine synthetase-encoding gene GLUTAMINE SYNTHETASE 1;4 (GLN1;4) were upregulated in WT plants under low N conditions compared to control N conditions (Figure 1). The expression levels of these genes were reduced in m123-1 plants compared to that in WT under low N conditions (Figure 1). The expression levels of NRT2.1 and GLN1;4 tend to be decreased in m123-1 plants compared to that in WT even in control N conditions although the differences were not statistically significant (Figure 1). We also analyzed the expression levels of the plastid-located glutamine synthetase-encoding gene GLUTAMINE SYNTHETASE (GS2) and NITRATE REDUCTASE 1 (NIA1), both of which are reportedly downregulated under low N conditions (Konishi and Yanagisawa 2011; Taira et al. 2004). Our results showed that expression levels of both GS2 and NIA1 were decreased to similar levels in m123-1 and WT plants under low N conditions (Figure 1).

Figure 1. Expression levels of nitrogen (N)-responsive marker genes in wild-type (WT) and nap1;1 nap1;2 nap1;3 triple mutant (m123-1) plants. Plants were grown on control (30 mM) N medium for 7 days and then on control (30 mM) or low (0.3 mM) N medium for 1 day. Expression levels of genes were first normalized relative to that of the 18S rRNA gene and then relative to the expression level in WT plants grown on control (30 mM) N medium. Results represent the mean±standard deviation (SD; n=4 biological replicates). Different letters indicate significant differences detected by two-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test (p<0.05).

N availability affects plant root architecture to optimize N acquisition efficiency (Kiba and Krapp 2016; López-Bucio et al. 2003). Additionally, in Arabidopsis, lateral root growth is promoted under N deficient conditions (Gruber et al. 2013). To further evaluate the function of NAP1 proteins under N deficient conditions, we analyzed the root growth phenotypes of WT and m123-1 seedlings grown first on control (30 mM) N medium for 6 days and then grown on control (30 mM) or low (0.3 mM) N medium for 4 days. The primary root length of WT plants was similar under both N conditions, but lateral root elongation was more promoted under the low N condition than under the control condition (Figure 2). Compared with the WT, m123-1 plants showed shorter primary root on both control and low N media (Figure 2A, B). Low N-induced lateral root elongation was significantly repressed in m123-1 plants (Figure 2A, C). The lateral root was shorter in m123-1 plants compared to that in WT even in control N conditions (Figure 2A, C). These results suggest that NAP1s are involved in root development and are required for the promotion of lateral root growth under N deficient conditions.

Figure 2. Root growth phenotype of WT and m123-1 mutant seedlings grown on different N media. (A) Plants were grown on control (30 mM) N medium for 6 days and then transferred to 30 mM or 0.3 mM N-containing medium. Photographs were taken 4 days after transfer, and root length was measured. Scale bar=1 cm. (B and C) Primary root length (B) and average lateral root length (C) of seedlings depicted in (A). Results represent mean±SD (n=8). Different letters indicate significant differences (two-way ANOVA and Tukey’s HSD test; p<0.05).

In addition, we monitored the progression of the N deficiency-induced leaf senescence in m123-1 plants. Under N deficient conditions, photosynthesis was downregulated, while nutrient recycling and remobilization were upregulated, resulting in the induction of leaf senescence (Avila-Ospina et al. 2014; Diaz et al. 2008). Since reduction in chlorophyll content is a typical marker of leaf senescence, we measured the chlorophyll content of the leaves of WT and m123-1 seedlings grown first on control (30 mM) N medium for 10 days and then on control (30 mM) or low (0.3 mM) N medium for 11 days. The leaves were harvested, frozen, and homogenized in 80% acetone to extract chlorophyll, which was quantified by measuring the absorbance at 663.6, 646.6, and 750 nm using a spectrophotometer (Aoyama et al. 2014). The total chlorophyll content was calculated from the sum of the content of chlorophyll a (Chl a) and chlorophyll b (Chl b) estimated by the following equations:

where A_663.6, A_646.6, and A₇₅₀ represent the absorbance at 663.6, 646.6, and 750 nm, respectively.

Leaves of WT plants showed yellowing and a significant reduction in chlorophyll contents under low N conditions compared with the control condition (Figure 3), as expected. By contrast, leaves of m123-1 plants exhibited delayed yellowing and contained more chlorophyll than WT plants (Figure 3). These results suggest that NAP1s are involved in the progression of leaf senescence under N deficient conditions.

Figure 3. Leaf senescence phenotype of WT and m123-1 mutant seedlings grown on different N media. (A) Photographs of plants grown on control (30 mM) N medium for 10 days and then on 30 mM or 0.3 mM N-containing medium for 11 days. (B) Total chlorophyll contents of plants shown in (A). Scale bar=1 cm. Results represent mean±SD (n=12). Different letters indicate significant differences (two-way ANOVA and Tukey’s HSD test; p<0.05).

Taken together, this study suggests that NAP1 proteins affect gene expression and plants growth under N deficient conditions in Arabidopsis. Note that NAP1s might be involved in regulating N transport and metabolism as well as root growth not only under N deficient condition but also control N conditions. Because NAP1 proteins are histone chaperones, it is expected that these proteins affect the expression levels of N-responsive genes via alterations of the nucleosome architecture to optimize the plants growth under N deficient conditions. Further investigation of the biochemical properties of NAP1 proteins and the identification of their target genes will provide new insights into the chromatin remodeling mechanisms employed by plants under N deficiency.

Conflict of interest

The authors declare no conflict of interest.

Author contribution

L.J. and T.S. designed the experiments. L.J., M.S., Y.L., H.M. and Y.F. performed the experiments. L.J., M.S., H.M., J.T. and T.S. analyzed the data. L.J. and T.S. wrote the article, and M.S., Y.F., Y.C., S.Y., J.Y. and J.T. edited it. All the authors approved the manuscript.

Description of Supplementary Files

Supplementary Table S1. Primers used in this study.