NLP7 is expressed in roots and shoots, mainly at the tip of the root and in the vascular tissue and in the stomata (Fig. 1a, b, c). NLP7-GFP is localized in the nucleus (Fig. 1d, e) in accordance with a function as transcription factor.
Figure 1. NLP7 promoter activity and NLP7 protein localisation. (a-c) GUS staining of roots carrying a pNLP7::GUS construct. (a) Root tip, (b) differentiated part of a root, (c) leaves mesophyll and guard cells. (d-e) confocal laser-scanning microscope observations of NLP7-GFP fusion proteins. (d) 35S::NLP7-GFP transformed Arabidopsis protoplast. (e) Differentiated part of Arabidopsis roots that carry a 35S::GFP-NLP7. Scale bars: a,b,e =150µm, c,d = 20µm. Plants were grown in vitro on 9mM nitrate medium for 10 days.
The functional analysis of NLP7 using a reverse genetic approach revealed that nlp7 loss of function mutants display several traits of a nitrogen starved plant despite growing on ample nitrogen.
- smaller shoot biomass and increased shoot/root ratio (an indicator for N starvation) (Fig. 2 a, b)
- increased lateral root density under ample N condition, which resembles plants grown under N starvation (Fig. 2 c)
- a global transcriptome pattern with similarity to transcriptome of N starved wild-type plants
Figure 2. Morphological phenotype of nlp7 mutants. (a) Plants grown in hydroponic culture for 21 days on a 1/2MS (1%sucrose) (b) Ratio of shoot biomass to root biomass (S/R) of plants shown in plants grown for 42 days in hydroponics. Black columns, plants grown on a 6 mM NO3- medium; white columns, plants grown on a 0.2 mM NO3- medium. Error bars represent the standard deviation from 3 biological repetitions of 3 plants each. (c) Plants grown for 12 days in vitro on 9 mM NO3- medium (+N) or on N-free medium (-N)
nlp7 mutant have a better tolerance to drought stress (Figure 3)
Figure 3. Water loss and drought resistance of the nlp7-1 mutant. Left photo; wild type and nlp7-1 mutant plants after 14 days without watering. Right photo; plants shown on the left after 5 days of water resupply. Twelve pots were examined per genotype and representative plants are shown.
Nitrate induction of N-assimilation genes is reduced in the nlp7 mutants (Fig. 4).
Figure 4. Nitrate induction of nitrate transporter and nitrate reductase genes in the roots of the nlp7 mutant. Plants grown in hydroponic culture for 21 days on a 6 mM NO3- medium were nitrogen starved for 5 days and then resupplied with 6 mM nitrate. Steady state amount of the high affinity nitrate transporter genes NRT2.1 and of the nitrate reductase genes NIA1 and NIA2 were studied by Q-RT-PCR. Results are given as a percentage of the EF1α gene (At5g60390) mRNA level. Black columns; Col8 plants. Grey columns; nlp7-1 plants. White columns, nlp7-2 plants. Error bars represent standard deviation from 3 biological repetitions of 4 plants each.
NLP7 is a master regulator of early nitrate induction
Using a ChIP-chip approach, we have shown that NLP7 binds to hundreds of genes, many of them involved in nitrate assimilation and signaling. Transcriptome analysis and trans-activation assays in protoplasts confirm the high hierarchy position of NLP7 in the nitrate signaling pathway (Figure 5).
Figure 5. Target genes of NLP7
Nitrate triggers the accumulation of NLP7 in the nucleus by inhibiting nuclear export
Figure 6. Addition of nitrate leads to a rapid accumulation of GFP-NLP7 in the nucleus, which can be mimicjed by the nuclear export inhibitor Leptomycin B.
We propose the following models for NLP7 action in planta (Fig 7 and 8).
|Figure 7. Schematic model of proposed NLP7 activation in plants.
Upon nitrate induction, NLP7 accumulates in the nucleus and leads to the transcriptional activation of hundreds of genes.
||Figure 8. Schematic model of proposed NLP7 action in plants.
Nitrate and nitrate starvation are perceived as signals by specific sensors like NRT1.1 and other still unknown receptors. The signal is then transmitted by a signal transduction pathway, including NLA and other unknown transducers towards transcription factors like ANR1 and NLP7 that trigger nitrate and nitrogen starvation-specific responses.
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