Abstract
Nitrogen (N) is a vital nutrient for crop growth and development which
influences both yield and quality. Melatonin (MT), a known enhancer of
abiotic stress tolerance, has been extensively studies; however, its
relationship with nutrient stress, particularly N, and the underlying
regulatory mechanisms of MT on N uptake remain unclear. In this study,
exogenous MT treatment was found to improve the tolerance of apple
plants to N deficiency. Apple plants overexpressing the MT biosynthetic
gene N-acetylserotonin methyltransferase 9 (MdASMT9 ) was used to
further investigated the effects of endogenous MT on low-N stress. The
overexpression of MdASMT9 improved the light harvesting and heat
transfer capability of apple plants, thereby mitigating the detrimental
effects of N deficiency on the photosynthetic system. Proteomic and
physiological data analyses indicated that MdASMT9 overexpression
enhanced the trichloroacetic acid (TCA) cycle and positively modulated
amino acid metabolism to counteract N-deficiency stress. Additionally,
both exogenous and endogenous MT promoted the transcription ofMdHY5 , which in turn bound to the MdNRT2.1 andMdNRT2.4 promoters and activated their expression. Notably,
MT-mediated promotion of MdNRT2.1 and MdNRT2.4 expression
in an MdHY5 -dependent manner, ultimately enhancing N absorption.
Taken together, these results may provide useful insights into the
relationship between MdASMT9 -mediated MT biosynthesis and N
uptake under N-deficiency conditions in apple plants.
Keywords: melatonin, MdASMT9 , nitrogen deficiency,MdHY5 , nitrate transporters proteins
INTRODUCTION
Nitrogen (N) is an essential macronutrient for plant growth and
development, serving as a main component of the nucleotides and
proteins. Its deficiency hinders the synthesis of nucleic acids,
hormones, chlorophyll, and other metabolites required for primary
metabolism (Yang et al. , 2015; Zrenner et al. , 2006). As a
result, plant productivity is largely dependent on N fertilization,
which is a limiting factor in agricultural production (Kaur et
al. , 2017). However, excessive N application leads to increased
costs, environmental pollution, and imperiled ecological conditions (Yu
et al. , 2019; Zhang et al. , 2015). Accordingly, enhancing
the N use efficiency of plants is of utmost importance for sustainable
crop production and effective agricultural research.
For plants, nitrate is one of the main forms of N available (Crawford
and Glass, 1998). In response to varying nitrate conditions, plants have
evolved the high-affinity nitrate transport system (HATS) and
low-affinity nitrate transport system (LATS) (Crawford and Glass, 1998;
Glass et al. , 1992). HATS operates at very low external nitrate
concentrations (below 1 mM) while LATS is engaged when the external
nitrate concentration exceeds 1 mM (Behl et al. , 1988; Siddiqi et
al. , 1990). Nitrate transporters proteins (NRTs) can be
categorized as NRT1 or NRT2 (Krapp et al. , 2014; Léran et
al. , 2014). Among the 53 NRT1 genes in Arabidopsis , NRT1.1
functions as a dual-affinity transporter, while the remaining members
are all low-affinity transporters (Liu et al. , 1999; Tsay et
al. , 2007). NRT2 proteins play a key role in response to low
nitrate conditions, with NRT2.1 being a major contributor to HATS
(Okamoto et al. , 2003; You et al. , 2022). However, NRT2.4
also exhibits a very high-affinity for nitrate and exerts dual effects
on plant shoots and roots under N-deficiency conditions (Kiba et
al. , 2012). In nrt2.4 mutants, reduced nitrate uptake and
nitrate content has been observed in shoot phloem exudates under low
external supply (Kiba et al. , 2012). Furthermore, AtNRT2.5 is
primarily expressed in nitrate-deprived plants roots as a complex with
AtNAR2.1 (Kotur and Glass, 2015).
Transcription factors (TFs) sever as regulators in plant signaling
networks and have been shown to regulate N uptake and transport. Dof1
promotes plant growth during N deficiency by regulating the expression
of genes related to carbon-skeleton production and by enhancing N
assimilation (Yanagisawa et al. , 2004). ANR1, a member of MADS
box, participates in the NRT1.1 signaling pathway and regulates lateral
root elongation in Arabidopsis (Remans et al. , 2006; Zhang
and Forde, 1998). In Arabidopsis , ELONGATED HYPOCOTYL5 (HY5), a
member of basic leucine zipper (bZIP) family, promotes nitrate
absorption by activating NRT2.1 as a shoot-to-root mobile signal
(Chen et al. , 2016). The overexpression of MdHY5 in apple
calli enhances the expression of MdNRT2.1 , MdNRT2.4 , andMdNRT2.7 , potentially aiding in the coordination of carbon (C)
and N acquisition (An et al. , 2017; Chen et al. , 2016).
Melatonin (MT, N-acetyl-5-methoxytryptamine) is a pleiotropic molecule
in living organisms responsible for a variety of effects. In plants, MT
is recognized as an antioxidant that confers tolerance to various types
of abiotic stress caused by drought (Liang et al. , 2018),
salinity (Li et al. , 2019; Yu et al. , 2018), cold (Li et
al. , 2018), and heavy metals (Yan et al. , 2019). Recent
studies have also revealed the positive influence of MT on N uptake.
Application of 1 μM MT has been shown to significantly enhance N
absorption and assimilation by increasing the activities of glutamine
synthetase and nitrate reductase under N-deficiency conditions (Qiao et
al. , 2019). Liang et al. (2018) found that MT application
increased 15N uptake, utilization, and accumulation by
upregulating genes involved in N absorption and metabolism during water
deficiency.
N-acetylserotonin methyltransferase (ASMT) is considered a key enzyme in
MT biosynthesis and is extremely important in regulating MT levels in
plants (Kang et al. , 2011; Liu et al. , 2017; Park et
al. , 2013). In our previous study, we characterizedMdASMT9 in apple plants, and demonstrated that its overexpression
increased MT levels and improved water-use efficiency in apple plants
(Zhou, Li, et al. , 2022). However, the specific functions of
endogenous MT in apple plants under low N conditions remain unclear. In
the present study, we used MdASMT9 -overexpressing (OE) apple
lines to reveal the function of endogenous MT in response to N
deficiency. Our findings indicate that the overexpression ofMdASMT9 enables plants to maintain higher photosynthetic capacity
and plays a positive role in the tolerance to N deficiency. Proteomic
and physiological data analyses indicate that MdASMT9overexpression enhances the TCA cycle and positively modulates amino
acid metabolism to alleviate N-deficiency stress. Furthermore, MT
promotes MdNRT2.1 and MdNRT2.4 expression in anMdHY5 -dependent manner, thereby improving N absorption.
MATERIALS AND METHODS