Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots

Abstract

Summary• Given the importance of nitrogen for plant growth and the environmental costs of intense fertilization, an understanding of the molecular mechanisms underlying the root adaptation to nitrogen fluctuations is a primary goal for the development of biotechnological tools for sustainable agriculture. This research aimed to identify the molecular factors involved in the response of maize roots to nitrate.• cDNA-amplified fragment length polymorphism was exploited for comprehensive transcript profiling of maize (Zea mays) seedling roots grown with varied nitrate availabilities; 336 primer combinations were tested and 661 differentially regulated transcripts were identified. The expression of selected genes was studied in depth through quantitative real-time polymerase chain reaction and in situ hybridization.• Over 50% of the genes identified responded to prolonged nitrate starvation and a few were identified as putatively involved in the early nitrate signaling mechanisms. Real-time results and in situ localization analyses demonstrated coregulated transcriptional patterns in root epidermal cells for genes putatively involved in nitric oxide synthesis ⁄ scavenging.• Our findings, in addition to strengthening already known mechanisms, revealed the existence of a new complex signaling framework in which brassinosteroids (BRI1), the module MKK2-MAPK6 and the fine regulation of nitric oxide homeostasis via the co-expression of synthetic (nitrate reductase) and scavenging (hemoglobin) components may play key functions in maize responses to nitrate.

Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L.

“…The application of exogenous brassinolide upregulates a large number of NRT genes in Arabidopsis seedling roots grown on both high and low nitrate plates (Kiba et al 2011). On the contrary, Trevisan et al (2011) reported that the BR receptor-like kinase BRI1 expression was down-regulated after 5 days of nitrate depletion in maize. Similarly, the BRI1 kinase inhibitor 1 gene BKI1, a negative regulator involved in the BR signaling pathway, was up-regulated under N deficiency in cucumber (Zhao et al 2015).…”

“…Total RNA (1 µl) was quantified using a Nanodrop 1000 (Thermo Scientific, Nanodrop Products, Wilmington, DE, USA). Finally, cDNA was synthesized from 500 ng of total RNA mixed with 1 µl of 10 µM oligo-dT, as described by Trevisan et al (2011).…”

Nitrogen and Stress

“…Locally, it affects root growth, seed dormancy, and fl owering time, but also has systemic effects on an organism-wide basis (Ho and Tsay 2010 ). For example, nitrate has been shown to induce genomewide changes in gene expression in model plants such as Arabidopsis (Wang et al 2003 ;Scheible et al 2004 ), rice (Lian et al 2006 ;Cai et al 2012 ), maize (Trevisan et al 2011 ), and tomato (Wang et al 2001 ), involving hundreds, if not thousands, of genes. They include various nitrate transporters, enzymes of nitrate assimilation, carbon and redox metabolism, several protein kinases, cytochrome families, transcription factors, etc.…”

NO signaling is a key component of the root growth response to nitrate inZea maysL.

“…Nevertheless, when an inhibitor of nitrate reductase activity (tungstate) or a nitric oxide scavenger (cPTIO) were supplied -(1mM) was demonstrated to be dependent on the control of nitric oxide (NO) homeostasis thank to the coordinate regulation of cytosolic nitrate reductase (NR) and non-symbiotic hemoglobins (nsHbs) (B). 67,68 The preferential localization and the strong transcriptional responsiveness of both NR and nsHbs in the transition zone of the apex straightened the hypothesis of a role of this root portion in translating the environmental stimuli in developmental response. 67,68 Because of the role of NO in several cytoskeleton-mediated processes in plants, [76][77][78][79][80][81][82][83] the actin-dependent endocytosis and the organization of the actin cytoskeleton are proposed as candidates in transducing the NO-dependent nitrate regulation of root elongation.…”

“…65,66 Furthermore, a recent study performed in maize provided evidences that NO is produced by nitrate reductase (NR) as an early response to nitrate supply and that the coordinated induction of non-symbiotic hemoglobins (nsHbs) could finely regulate the NO steady-state. 67,68 Both nitric oxide biosynthesis and gene regulation were preferentially accomplished by cells of the transition zone of roots, which would seem the most nitrate responsive portion of maize root. 68 NsHbs play important roles in plant physiology by regulating a number of downstream physiological events involved in plant developmental processes and stress responses, also interacting with many hormonal signaling (for a review see refs 69,70).…”