Riliang Gu

Allosteric Regulation of Transport Activity by Heterotrimerization of Arabidopsis Ammonium Transporter Complexes in Vivo

In plants, AMT-type ammonium transporters are posttranslationally regulated by phosphorylation. This study provides evidence that allosteric regulation is functional in planta and that C-terminal phosphorylation mediates trans-inactivation also in heteromeric AMT complexes containing different AMT isoforms. Ammonium acquisition by plant roots is mediated by AMMONIUM TRANSPORTERs (AMTs), ubiquitous membrane proteins with essential roles in nitrogen nutrition in all organisms. In microbial and plant cells, ammonium transport activity is controlled by ammonium-triggered feedback inhibition to prevent cellular ammonium toxicity. Data from heterologous expression in yeast indicate that oligomerization of plant AMTs is critical for allosteric regulation of transport activity, in which the conserved cytosolic C terminus functions as a trans-activator. Employing the coexpressed transporters AMT1;1 and AMT1;3 from Arabidopsis thaliana as a model, we show here that these two isoforms form functional homo- and heterotrimers in yeast and plant roots and that AMT1;3 carrying a phosphomimic residue in its C terminus regulates both homo- and heterotrimers in a dominant-negative fashion in vivo. 15NH4+ influx studies further indicate that allosteric inhibition represses ammonium transport activity in roots of transgenic Arabidopsis expressing a phosphomimic mutant together with functional AMT1;3 or AMT1;1. Our study demonstrates in planta a regulatory role in transport activity of heterooligomerization of transporter isoforms, which may enhance their versatility for signal exchange in response to environmental triggers.

Characterization of AMT-Mediated High-Affinity Ammonium Uptake in Roots of Maize (Zea mays L.)

High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 μM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

A nonsymbiotic hemoglobin gene from maize, ZmHb, is involved in response to submergence, high-salt and osmotic stresses

Nonsymbiotic hemoglobins (nsHbs) are involved in a variety of cellular processes in plants. Previous studies on nsHbs suggest their function in response to hypoxic stress. Here, we report on the cloning and characterization of a maize nsHb gene (ZmHb) and its 5′ flanking sequences from maize inbred Zong31. Southern analysis suggests that the ZmHb gene is present in two copies or a low number of copies in the maize genome. Expression analysis by Northern blot shows that ZmHb mRNA levels in maize seedlings are induced by high-salt and osmotic stresses in addition to hypoxic stress. Promoter-GUS analysis has revealed that ZmHb promoter-driven GUS activity is localized to root tips and vascular tissues. Moreover, it is under similar patterns of regulation based on mRNA levels under the above environmental stress conditions. Ectopic expression of ZmHb in transgenic tobacco has enhanced plant tolerance to submergence, salinity and osmotic stresses. These results indicate that expression of ZmHb at transcript levels is regulated under multiple stress conditions in maize roots, suggesting an important role of ZmHb in plant stress tolerance.

A genetic relationship between nitrogen use efficiency and seedling root traits in maize as revealed by QTL analysis

Highlight This research determined the significant genetic and phenotypic relationships between seedling root traits and nitrogen use efficiency (NUE), and further identified five QTL clusters for improving NUE in maize.

Isolation and characterization of three maize aquaporin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 involved in urea transport.

Urea-based nitrogen fertilizer was widely utilized in maize production, but transporters involved in urea uptake, translocation and cellular homeostasis have not been identified. Here, we isolated three maize aquapoin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4, from a cDNA library by heterogeneous complementation of a urea uptake-defective yeast. ZmNIP2;1 and ZmNIP2;4 belonged to the nodulin 26-like intrinsic proteins (NIPs) localized at plasma membrane, and ZmTIP4;4 belonged to the tonoplast intrinsic protein (TIPs) at vacuolar membrane. Quantitative RT-PCR revealed that ZmNIP2;1 was expressed constitutively in various organs while ZmNIP2;4 and ZmTIP4;4 transcripts were abundant in reproductive organs and roots. Expression of ZmTIP4;4 was significantly increased in roots and expanded leaves under nitrogen starvation, while those of ZmNIP2;1 and ZmNIP2;4 remained unaffected. Functions of maize aquapoin genes in urea transport together with their distinct expression manners suggested that they might play diverse roles on urea uptake and translocation, or equilibrating urea concentration across tonoplast.

Comparative expression and phylogenetic analysis of maize cytokinin dehydrogenase/oxidase (CKX) gene family.

Cytokinin dehydrogenase (CKX) degrades the cytokinin hormone in plants and plays an important role in cytokinin regulatory processes. CKX proteins are encoded by a multigene family with a varying number of members. In this study, 13 maize CKX sequences were collected in which ten transcripts were confirmed by RT-PCR. The tissue- and cytokinin-dependent expression studies indicated that ZmCKX genes exhibit a variety of expression patterns, suggesting diverse functions. Besides 13 maize CKXs, 7 Arabidopsis, 9 poplar, and 11 rice CKX proteins were further used to construct a phylogenetic tree. The CKX members were assigned to six groups, and the intron/exon structures, sequence motifs, and protein properties were conserved within groups. The genome distribution of CKXs supports that segmental duplication contributes to the expansion of the CKX gene family. By quantitative RT–PCR analysis of maize members and digital Northern analysis of Arabidopsis, poplar, and rice members for their tissue expression patterns, highly correlative expression profiles of CKX genes were found among some of the orthologs, whereas different expression manners were found between some of the paralogs. These results suggest functional conservation within each group of the CKX family and provide a clue for transfer of a gene function from one species to the other and further contribute to uncovering the role of CKX genes in planta.

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco.

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA3, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.

Isolation and Analysis of Cold Stress Inducible Genes in Zea mays by Suppression Subtractive Hybridization and cDNA Macroarray

In order to understand the molecular and cellular mechanisms underlying cold stress conditions (4°C) in maize seedlings, a forward subtractive cDNA library was constructed using the suppression subtractive hybridization (SSH) technique. Through the “Virtual” Northern blot analysis, 893 positive clones were screened from a total 1,200 clones in the subtractive cDNA library. After sequencing 528 randomly chosen cDNA clones, 213 uniquely expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. Based on a list of functional Arabidopsis protein categories, the ESTs with significant protein similarity were sorted into ten functional categories. A cDNA macroarray containing the 213 unique ESTs was used to monitor the spatial and temporal distribution of gene expression in maize seedlings during cold stress. The results showed that 118 ESTs were induced by cold-stress in maize seedlings and 66 ESTs identified in the leaves and 89 ESTs in the roots. Hierarchical cluster analysis indicated that the expression profiles of cold stress inducible ESTs in the leaves were different from that observed in the roots. Moreover, some induced genes were related to sugar synthesis and reestablishment of high rates of photosynthesis. In addition, Northern blot analysis validated well the cDNA macroarray data.

Use of genotype-environment interactions to elucidate the pattern of maize root plasticity to nitrogen deficiency

Maize (Zea mays L.) root morphology exhibits a high degree of phenotypic plasticity to nitrogen (N) deficiency, but the underlying genetic architecture remains to be investigated. Using an advanced BC4 F3 population, we investigated the root growth plasticity under two contrasted N levels and identified the quantitative trait loci (QTLs) with QTL-environment (Q × E) interaction effects. Principal components analysis (PCA) on changes of root traits to N deficiency (ΔLN-HN) showed that root length and biomass contributed for 45.8% in the same magnitude and direction on the first PC, while root traits scattered highly on PC2 and PC3. Hierarchical cluster analysis on traits for ΔLN-HN further assigned the BC4 F3 lines into six groups, in which the special phenotypic responses to N deficiency was presented. These results revealed the complicated root plasticity of maize in response to N deficiency that can be caused by genotype-environment (G × E) interactions. Furthermore, QTL mapping using a multi-environment analysis identified 35 QTLs for root traits. Nine of these QTLs exhibited significant Q × E interaction effects. Taken together, our findings contribute to understanding the phenotypic and genotypic pattern of root plasticity to N deficiency, which will be useful for developing maize tolerance cultivars to N deficiency.

Evolution and expression analysis of the β-glucosidase (GLU) encoding gene subfamily in maize

The maize β-glucosidase (ZmGLU1) hydrolyzes cytokinin-conjugates for releasing active cytokinins and thus plays important roles in cytokinin regulatory processes. ZmGLU1 belongs to glycosyl hydrolases 1 (GH1) gene family with a large number of members, and the gene function of other homologs remains to be investigated. In this study, 47 Arabidopsis, 34 rice, 31 brachypodium, 28 sorghum and 26 maize GH1 protein sequences were collected and subsequently used to construct a phylogenetic tree by Neighbor-Joining method. ZmGLU1 together with its 7 paralogs and 4 sorghum homologs were assigned into a distinct group (named GLU subfamily) with far evolutionary distance to other GH1 members. None of the Arabidopsis, rice and brachypodium gene falling into this group indicated a recent evolutionary emergence of GLU subfamily in some Poaceae plants after the divergence of Poaceae species. Phylogeny and comparative genome analysis revealed that GLU subfamily members of maize and sorghum evolved from a common ancestor, and expanded independently in each species by several duplications after maize-sorghum split. Ka/Ks analysis showed that purifying selection played important roles in maintenance of similar functions among the maize GLU paralogs. In addition, the similar protein properties and cytokinin-dependent gene expressions further suggested the similar functions of ZmGLUs in cytokinin activation. However, the organ-dependent expression of ZmGLUs exhibited diverse patterns, which might contribute to their diverse roles in cytokinin homeostasis. Taken together, this work put new insights into the evolution and expression of ZmGLU genes, and provided the foundation for future functional investigations.