ZmNRT1.1B (ZmNPF6.6) determines nitrogen use efficiency via regulation of nitrate transport and signalling in maize.

Nitrate (NO3 - ) is crucial for optimal plant growth and development and often limits crop productivity under low availability. In comparison with model plant Arabidopsis, the molecular mechanisms underlying NO3 - acquisition and utilization remain largely unclear in maize. In particular, only a few genes have been exploited to improve nitrogen use efficiency (NUE). Here, we demonstrated that NO3 - -inducible ZmNRT1.1B (ZmNPF6.6) positively regulated NO3 - -dependent growth and NUE in maize. We showed that the tandem duplicated proteoform ZmNRT1.1C is irrelevant to maize seedling growth under NO3 - supply; however, the loss of function of ZmNRT1.1B significantly weakened plant growth under adequate NO3 - supply under both hydroponic and field conditions. The 15 N-labelled NO3 - absorption assay indicated that ZmNRT1.1B mediated the high-affinity NO3 - -transport and root-to-shoot NO3 - translocation. Transcriptome analysis further showed, upon NO3 - supply, ZmNRT1.1B promotes cytoplasmic-to-nuclear shuttling of ZmNLP3.1 (ZmNLP8), which co-regulates the expression of genes involved in NO3 - response, cytokinin biosynthesis and carbon metabolism. Remarkably, overexpression of ZmNRT1.1B in modern maize hybrids improved grain yield under N-limiting fields. Taken together, our study revealed a crucial role of ZmNRT1.1B in high-affinity NO3 - transport and signalling and offers valuable genetic resource for breeding N use efficient high-yield cultivars.

Involvement of a truncated MADS-box transcription factor ZmTMM1 in root nitrate foraging.

Plants can develop root systems with distinct anatomical features and morphological plasticity to forage nutrients distributed heterogeneously in soils. Lateral root proliferation is a typical nutrient-foraging response to a local supply of nitrate, which has been investigated across many plant species. However, the underlying mechanism in maize roots remains largely unknown. Here, we report on identification of a maize truncated MIKC-type MADS-box transcription factor (ZmTMM1) lacking K- and C-domains, expressed preferentially in the lateral root branching zone and induced by the localized supply of nitrate. ZmTMM1 belongs to the AGL17-like MADS-box transcription factor family that contains orthologs of ANR1, a key regulator for root nitrate foraging in Arabidopsis. Ectopic overexpression of ZmTMM1 recovers the defective growth of lateral roots in the Arabidopsis anr1 agl21 double mutant. The local activation of glucocorticoid receptor fusion proteins for ZmTMM1 and an artificially truncated form of AtANR1 without the K- and C-domains stimulates the lateral root growth of the Arabidopsis anr1 agl21 mutant, providing evidence that ZmTMM1 encodes a functional MADS-box that modulates lateral root development. However, no phenotype was observed in ZmTMM1-RNAi transgenic maize lines, suggesting a possible genetic redundancy of ZmTMM1 with other AGL17-like genes in maize. A comparative genome analysis further suggests that a nitrate-inducible transcriptional regulation is probably conserved in both truncated and non-truncated forms of ZmTMM1-like MADS-box transcription factors found in grass species.

Increased biomass accumulation in maize grown in mixed nitrogen supply is mediated by auxin synthesis.

The use of mixed nitrate and ammonium as a nitrogen source can improve plant growth. Here, we used metabolomics and transcriptomics to study the underlying mechanisms. Maize plants were grown hydroponically in the presence of three forms of nitrogen (nitrate alone, 75%/25% nitrate/ammonium, and ammonium alone). Plants grown with mixed nitrogen had a higher photosynthetic rate than those supplied only with nitrate, and had the highest leaf area and shoot and root biomass among the three nitrogen treatments. In shoot and root, the concentration of nitrogenous compounds (ammonium, glutamine, and asparagine) and carbohydrates (sucrose, glucose, and fructose) in plants with a mixed nitrogen supply was higher than that with nitrate supply, but lower than that with ammonium supply. The activity of the related enzymes (glutamate synthase, asparagine synthase, phosphoenolpyruvate carboxylase, invertase, and ADP-glucose pyrophosphorylase) changed accordingly. Specifically, the mixed nitrogen source enhanced auxin synthesis via the shikimic acid pathway, as indicated by the higher levels of phosphoenolpyruvate and tryptophan compared with the other two treatments. The expression of corresponding genes involving auxin synthesis and response was up-regulated. Supply of only ammonium resulted in high levels of glutamine and asparagine, starch, and trehalose hexaphosphate. We conclude that, in addition to increased photosynthesis, mixed nitrogen supply enhances leaf growth via increasing auxin synthesis to build a large sink for carbon and nitrogen utilization, which, in turn, facilitates further carbon assimilation and nitrogen uptake.

Phylogenetic, expression and functional characterizations of the maize NLP transcription factor family reveal a role in nitrate assimilation and signaling.

Although nitrate represents an important nitrogen (N) source for maize, a major crop of dryland areas, the molecular mechanisms of nitrate uptake and assimilation remain poorly understood. Here, we identified nine maize NIN-like protein (ZmNLP) genes and analyzed the function of one member, ZmNLP3.1, in nitrate nutrition and signaling. The NLP family genes were clustered into three clades in a phylogenic tree. Comparative genomic analysis showed that most ZmNLP genes had collinear relationships to the corresponding NLPs in rice, and that the expansion of the ZmNLP family resulted from segmental duplications in the maize genome. Quantitative PCR analysis revealed the expression of ZmNLP2.1, ZmNLP2.2, ZmNLP3.1, ZmNLP3.2, ZmNLP3.3, and ZmNLP3.4 was induced by nitrate in maize roots. The function of ZmNLP3.1 was investigated by overexpressing it in the Arabidopsis nlp7-1 mutant, which is defective in the AtNLP7 gene for nitrate signaling and assimilation. Ectopic expression of ZmNLP3.1 restored the N-deficient phenotypes of nlp7-1 under nitrate-replete conditions in terms of shoot biomass, root morphology and nitrate assimilation. Furthermore, the nitrate induction of NRT2.1, NIA1, and NiR1 gene expression was recovered in the 35S::ZmNLP3.1/nlp7-1 transgenic lines, indicating that ZmNLP3.1 plays essential roles in nitrate signaling. Taken together, these results suggest that ZmNLP3.1 plays an essential role in regulating nitrate signaling and assimilation processes, and represents a valuable candidate for developing transgenic maize cultivars with high N-use efficiency.

Comparative Analysis of Root Traits and the Associated QTLs for Maize Seedlings Grown in Paper Roll, Hydroponics and Vermiculite Culture System.

Root system architecture (RSA) plays an important role in the acquisition of both nitrogen (N) and phosphorus (P) from the environment. Currently RSA is rarely considered as criteria for selection to improve nutrient uptake efficiency in crop breeding. Under field conditions roots can be greatly influenced by uncontrolled environment factors. Therefore, it is necessary to develop fast selection methods for evaluating root traits of young seedlings in the lab which can then be related to high nutrient efficiency of adult plants in the field. Here, a maize recombination inbred line (RILs) population was used to compare the genetic relationship between RSA and nitrogen and phosphorous efficiency traits. The phenotypes of eight RSA-related traits were evaluated in young seedlings using three different growth systems (i.e., paper roll, hydroponics and vermiculite), and then subjected to correlation analysis with N efficiency and P efficiency related traits measured under field conditions. Quantitative trait loci (QTL) of RSA were determined and QTL co-localizations across different growth systems were further analyzed. Phenotypic associations were observed for most of RSA traits among all three culture systems. RSA-related traits in hydroponics and vermiculite weakly correlated with Nitrogen (NupE) uptake efficiency (r = 0.17-0.31) and Phosphorus (PupE) uptake efficiency (r = 0.22-0.34). This correlation was not found in the paper roll growth system. A total of 14 QTLs for RSA were identified in paper rolls, 18 in hydroponics, and 14 in vermiculite. Co-localization of QTLs for RSA traits were identified in six chromosome regions of bin 1.04/1.05, 1.06, 2.04/2.05, 3.04, 4.05, and 5.04/5.05. The results suggest the problem of using the phenotype from one growth system to predict those in another growth system. Assessing RSA traits at the seedling stage using either hydroponics or a vermiculite system appears better suited than the paper roll system as an important index to accelerate the selection of high N and P efficient genotypes for maize breeding programs.

Arabidopsis formin3 directs the formation of actin cables and polarized growth in pollen tubes.

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785-amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.

Heterosis in root development and differential gene expression between hybrids and their parental inbreds in wheat (Triticum aestivum L.).

In spite of commercial use of heterosis in agriculture, the molecular basis of heterosis is poorly understood. In this study, heterosis was estimated for eight root traits in 20 wheat hybrids derived from a NC Design II mating scheme. Positive mid-parent heterosis was detected in 96 of 160 hybrid-trait combinations, and positive high-parent heterosis was detected in 79 of 160 hybrid-trait combinations. Improved differential display was used to analyze alterations in gene expression between hybrids and their parents in roots at the jointing stage. More than 990 fragments were repeatedly displayed, among which 27.52% were differentially expressed between hybrids and their parents. Four differential expression patterns were observed. Thirty differentially expressed cDNA fragments and three genes with open reading frames were cloned, and their expression patterns were confirmed by reverse-northern blot and semi-quantitative RT-PCR analysis, respectively. We concluded that these differentially expressed genes, though mostly with unknown function, could play important roles for hybrids to demonstrate heterosis in root system traits.

Relationship between differences of gene expression in early developing seeds of hybrid versus parents and heterosis in wheat.

In order to understand molecular basis of heterosis, mRNA differential display was used to analyze the differences in gene expression between seeds of 18 reciprocal hybrids and their 6 parents at 6th day after pollination. The relationship between gene expression patterns and heterosis was determined. Only bands that can be repeated in duplicate PCR were used for analysis so as to reduce false positive bands. Among the total of 2,025 bands displayed, 1,386 bands (68.43%) were reproducible. Eight patterns (fifteen kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in both hybrids and one parent (two kinds); (3) bands detected in parents and one hybrid (two kinds); (4) bands displayed in only one hybrid (two kinds); (5) bands revealed in one hybrid and its corresponding female (two kinds) or male (two kinds) parent; (6) bands visualized in only both hybrids (one kind); (7) bands occurring in only parents (one kind); (8) bands observed in parents and both hybrids (one kind). Our results indicate that differences of gene expression between hybrids and their parents are very obvious. The percentages of bands observed in only both hybrids and in only both parents are lower. The analysis shows that bands observed in parents and both hybrids are not correlated with all nine agronomic traits, which indicates differentially expressed genes are mainly responsible for the observed heterosis. At least one pattern of differential gene expression is significantly correlated with nine agronomic traits. Silenced bands in hybrid (which include bands occurring in only one parent, bands detected in both parents and one hybrid and bands in only parents) and bands present in one hybrid and its corresponding female or male parent are likely to play important roles in heterosis. These results suggest that early seed development could be closely related to heterosis.