| Plant roots have evolved morphological plasticity and functional adaptability to exploit heterogeneously distributed mineral nutrients from soil. Post-embryonic branching of lateral roots senses the external signal and regulates internally to reconstract root system architecture targeting on the improvements of the ability of nutrients acquisition and environmental adaptability. Nitrate distribution and availability in the soil are key factors controlling lateral root elongation and initiation. Differing from model plant Arabidopsis, the molecular mechanisms underlying heterogeneously distributed nitrate-dependent root response is poorly understood because of complex maize (Zea mays. L) root system architecture and morphology. Maize is a staple food and energy crop all over the world. Excessive nitrogen (N) application in maize production results in low nitrogen use efficiency (NUE) without yield benefits and can have profound environmental consequences during its production. Taking advantage of strategies on plant physiology, molecular biology and bioinformatics, we investigated the physiological, molecular and genetic mechanisms of local high nitrate triggering lateral root formation in maize. By integrated analyses of published data in the past 50 years and 2-year field experiments validation, we demonstrated the positive relationship between the NUE and root system architecture and its distribution. The main results were as follows:(1) Root morphological responses to local high nitrate supply among seedling primary root, shoot-borne roots initiated from the 2nd/5th/7th nodes were characterized in maize. Local high nitrate supply only increased lateral root length of the treated primary roots of seedlings, shoot-borne roots initiated from the 2nd and 5th nodes, but increased both lateral root length and density of the shoot-borne roots initiated from the 7th node of silking maize plants. The 2nd-order lateral roots on shoot-borne roots displayed a more plastic response to local high nitrate than the 1st-order lateral roots. Length and density of the lateral roots of shoot-borne roots initiated from the 7th node displayed more plastic response than the other whorls of shoot-borne roots. The number, diameter and the number of metaxylem vessels of shoot-borne roots initiated from different nodes increased with the prolonged growth period, which showed a positive relationship with shoot growth and N accumulation. Local high nitrate supply significantly inhibited the root growth outside of the nitrate-rich patches at maize seedlings.(2) By dissecting the physiological acquisition of primary root and silking shoot-borne root in response to local high nitrate supply, it was found that local high nitrate supply significantly increased plant total N content but not nitrate influx rate of the treated roots, when expressed as per unit of root length; and inhibited the corresponding genes expression of ZmNrt2.1 and ZmNrt2.2. It was concluded that local high nitrate supply increased plant total N content substantially by morphological plasticity, rather than by physiological alterations.(3) By histological and histochemical analyses on the silking shoot-borne roots of maize, it was found that local high nitrate supply triggered lateral root initiation by increasing the frequency of divisions of pericycle cells adjacent to phloem poles. By peeling off the stele tissue from the cortex tissue, combining stele-specific RNA sequencing (RNA-Seq) with dynamic gene expression profiling, it was revealed that B-type cell cycle activators (cyclin-dependent kinases B, CDKB) were up-regulated, whereas repressors during cell cycle(Kip-related proteins, KRP) were down-regulated in the pericycle. By adopting DR5::RFP transgenic lines and determination of auxin concentration using Ultraperformance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry, it was found that the resulted DR5-red fluorescent protein-marked auxin response maxima at the phloem poles was preceded by the free auxin from the root meristem via lateral root cap to the lateral root initiation zone. Laser Capture Microdissection (LCM) coupling with qRT-PCR further indicated that local high nitrate supply regulated ZmPIN9-mediated auxin efflux from the phloem poles and endodermis to the neighbor pericycle cells prior to lateral root initiation.(4) Morphological and histochemical analyses revealed that local high nitrate supply failed to induce pericycle cell divisions and subsequent initiation of seedling primary root, seminal roots and crown roots. Further dissections into the differences on the transcriptional signature of pericycle cells adjacent to phloem poles of the corresponding three root types and silking shoot-borne roots by coupling Laser Capture Microdissection with RNA sequencing (LCM-RNA-Seq), it was found that the transcriptomes similarity of silking shoot-borne roots substantially deviated from the other three root types when exposing the different root types to homogeneous low nitrate or local high nitrate supplies. The variations of transcriptomes induced by local high nitrate supply in silking shoot-borne roots was smaller than the corresponding differences from the transcriptomes of the other three root types. Total 3,313 active genes were detected in the pericycle cells adjacent to the phloem poles of silking shoot-borne roots, which was significantly higher than those detected in the other three root types. By conducting the clustering analysis to compare the trends of gene expression profile under homogeneous low or local high nitrate supply, it was shown that 30-40% genes representing significant gene expression transitions from stable expression in seedling primary root, seminal roots and crown roots to significant higher induced expression in silking shoot-borne roots. Further analysis on the transcriptional response to the local high nitrate supply in the pericycle cells among the four root types revealed 3,046 nitrate-responsive genes in silking shoot-borne roots,589 genes specifically in seminal root and 11 genes exclusively in crown roots, but no genes were found in primary root.(5) Root dry weight, root/shoot dry weight ratio, and their relationship with NUE of maize grown in the field in China and western countries were compared and analyzed using the data from 106 studies published since 1959. Detailed analysis revealed that the root dry weight and root/shoot dry weight ratio of maize cultivars bred in China was smaller than those bred in western countries, which were not derived from variations in the environmental factors (climate, geography, and stress factors). It was found that NUE was positively correlated with root/shoot dry weight ratio. We then validated this conclusion by conducting 2-year field trials and demonstrated that root size, root/shoot dry weight ratio and its spatial distribution influenced significantly NUE in maize production.By systemic tracking on morphological, physiological, histological and histochemical differences on the lateral root initiation in response to local high nitrate supply among diverse root types in maize, the distinct characteristics on the increase of lateral root density in silking shoot-borne roots were revealed. By integrated modern molecular biological technologies such as RNA-Seq combining with dynamic gene expression profiling, auxin efflux and cell cycle regulation mechanism in response to local high nitrate supply was found during lateral root initiation of silking shoot-borne roots in maize. Using optimized LCM-RNA-Seq technique coupling with bioinformatical strategies, the divergent mechanisms on lateral root initiation were profiled among root types at the cell level in maize. Integration of meta-analysis on published data and field validation demonstrated biological potential of root growth and spatial distribution on increasing NUE in maize production. |
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