A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation

Abundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.     

Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.     

Expression of miRNAs involved in phosphate homeostasis and senescence is altered in glyphosate-treated maize

Glyphosate is a systemic, nonselective and most widely used herbicide in the world. The introduction of glyphosate-resistant crops in the mid-1990s resulted in a dramatic increase in the use of glyphosate herbicide making it most widely used herbicide in the world. The average maize yield loss in the field caused by pests is around 20 % but in many regions it is much higher. It is now clear that glyphosate causes broader range of physiological alterations than previously assumed and some plants gain higher level of resistance to glyphosate without the need to use genetic engineering methods. To understand the mechanisms of such heightened resistance we must first know the processes mediating the plants’ death in response to glyphosate treatment. Here, we show that 12 miRNAs, belonging to miR167, miR396, miR159, miR156, miR169, miR444 and miR827 families, are significantly upregulated, and one, miR166, downregulated following glyphosate treatment. These miRNAs have been previously shown to be involved in abiotic stress responses and implicated in senescence. Strikingly, two of the induced miRNAs, miR444 and miR827, have been shown to regulate phosphate transport pathways, which seem to be common for Pi and glyphosate uptake.     

Regulatory network of microRNA399 and PHO2 by systemic signaling

Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme 24. Arabidopsis (Arabidopsis thaliana) plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots. The association of Pi translocation and coexpression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. In this study, we used reciprocal grafting between wild-type and miR399-overexpressing transgenic plants to dissect the systemic roles of miR399 and PHO2. Arabidopsis rootstocks overexpressing miR399 showed high accumulation of Pi in the wild-type scions because of reduced PHO2 expression in the rootstocks. Although miR399 precursors or expression was not detected, we found a small but substantial amount of mature miR399 in the wild-type rootstocks grafted with transgenic scions, which indicates the movement of miR399 from shoots to roots. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also discovered in grafted tobacco (Nicotiana benthamiana) plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 small interfering RNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency is discussed.     

Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.     

Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.     

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.     

Role of hydrogenases 1 and 2 in the hydrogen dependent nitrate respiration by Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to be dependent on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing the nitrate-dependent hydrogen consumption than is hydrogenase 1.