Abstract
Integrating carbon (C), nitrogen (N) and sulfur (S) metabolism is essential for the growth and development of living organisms. MicroRNAs (miRNAs) play key roles in regulating nutrient metabolism in plants. However, how plant miRNAs mediate crosstalk between different nutrient metabolic pathways is unclear. In this study, deep sequencing of Arabidopsis thaliana small RNAs was used to reveal miRNAs that were differentially expressed in response to C, N, or S deficiency. Comparative analysis revealed that the targets of the differentially expressed miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin signal transduction, nutrient homeostasis and regulation of development. C, N and S deficiency specifically induced miR169b/c, miR826 and miR395, respectively. In contrast, miR167, miR172, miR397, miR398, miR399, miR408, miR775, miR827, miR841, miR857 and miR2111 are commonly suppressed by C, N and S deficiency. In particular, the miRNAs that are induced specifically by a certain nutrient deficiency are often suppressed by other nutrient deficiencies. Further investigation indicated that the modulation of nutrient-responsive miRNA abundance affects the adaptation of plants to nutrient starvation conditions. This study revealed that miRNAs function as important regulatory nodes of different nutrient metabolic pathways.
Similar content being viewed by others
Introduction
Plants often grow in soil that contains very low concentrations of macronutrients or suffer from an unbalanced supply of macronutrients. To adapt and grow in nutrient-deprived environments, plants have developed strategies to cope with different nutrient ion limitations. When plants encounter starvation of a certain mineral nutrient, the metabolism of other nutrients will be adjusted to maintain proper growth and development. Previous studies suggested that the balance between carbon (C) and nitrogen (N), rather than one single metabolite, affects global gene expression. In addition, cellular C and N metabolism is tightly coordinated for C-N-containing metabolite synthesis at the biochemical level and for long-distance sensing and signaling of the C/N balance1. Microarray-base transcript analysis suggested that both N starvation2 and N-resupply after starvation3,4 affect many genes involved in C or S metabolism. Direct testing of the interactive effects of C/N on genome-wide gene expression supported the view that more than half of Arabidopsis’ genes are either induced or suppressed by C alone, N alone, or C-N combinations5,6,7.
After entering the cell, sulfate is activated and reduced and then incorporated into cysteine. The formation of cysteine involves the conjugation of sulfide with O-acetylserine (OAS), which is produced through the acetylation of serine. Provision of serine depends on adequate C and N metabolism and this convergence node of the assimilation pathways represents an opportunity for the coordination of S assimilation with C and N metabolism. When plants were fed with sucrose or glucose, the key enzyme of sulfate reduction, APS reductase was stimulated8,9,10. The sulfate transporters, SULTR1;1 and SULTR1;2, are mainly responsible for sulfate uptake from soil to the roots11. The mRNA levels of SULTR1;1 and SULTR1;2 were substantially increased in plants grown with sucrose under S-deficient (–S) conditions12. In contrast, removing sucrose from the media considerably attenuated the induction of SULTR1;1 and SULTR1;2 by –S13. These results suggested that the assimilation and transport of sulfate are not only regulated by the S status, but also controlled by the fluctuation of the C status. In addition to the influences of the C status, the expression of SULTR1;1 and SULTR1;2 was also significantly regulated by the N status. Similar to the contribution of C starvation, the reduction of the N supply significantly suppressed the induction of SULTR1;1 and SULTR1;2 by S depletion13. In contrast, the sulfate deprivation disrupted N metabolism, resulting in high levels of nitrate in leaves14.
Amino acids are necessary for the synthesis of proteins, which maintains the whole life cycle of plants. N and C are essential for the synthesis of amino acids and S-containing Met is required for protein synthesis. Therefore, the coordination of C, N and S metabolism ensures the fitness and propagation of plants. In fact, in addition to the interaction between the C, N and S metabolisms, other nutrient metabolisms also interact with C, N and S assimilation, such as phosphorus (P) and potassium (K)15,16,17,18,19. Despite genome-wide gene expression analyses, the mechanism underlying the crosstalk among C, N and S metabolic pathways is unclear, as is the crosstalk of C/N/S with other nutrient metabolic pathways.
Plant microRNAs (miRNAs) are a particular class of small RNAs that mainly participate in gene regulation at the post-transcription level. miRNAs are processed from endogenous mRNA with a stem-loop structure by Dicer-like proteins, which are further incorporated into RISC (RNA induced Silencing Complex) and then recruited to their target mRNAs by complementary base-pair. At last, miRNAs mediate the cleavage or (and) translation repression of target mRNAs20. Several miRNAs involved in nutrient metabolic pathways have been characterized. For example, miR395 and miR399 mediate the regulation of sulfate and phosphate homeostasis, respectively21,22. Copper (Cu) starvation induces miR397, miR398, miR408 and miR857 and mediates the downregulation of copper proteins23,24. miR169 and miR826 oppositely regulate N starvation response25,26. miR827 plays pivotal roles in regulating phosphate homeostasis in plants in a nitrate-dependent fashion27. In addition, deep sequencing identified some nutrient-responsive miRNAs, such as miR778, miR828 and miR2111, which were induced during phosphate limitation28,29.
To cope with nutrient-deprived environments, plants must sense changes in the external and internal mineral nutrient concentrations and adjust their nutrient metabolisms to meet the demands of plant growth. miRNAs are enriched in the vascular tissues30,31 where the translocation and distribution of nutrients occurs. miR399 is a long-distance signal for phosphate homeostasis32. Therefore, nutrient-responsive miRNAs might play crucial roles in modulating nutrient starvation responses and crosstalk among nutrient metabolic pathways. To date, how plant miRNAs mediate the interaction and crosstalk among different nutrient metabolisms remains unknown. This study systemically analyzed the expression of miRNAs under C, N, or S starvation conditions. Some miRNAs are specifically responsive to a specific nutrient deficiency and others are regulated differentially by different nutrient deficiency conditions. This study confirmed that a large number of miRNAs are responsive to nutrient deficiencies and some of them mediate the interaction between different nutrient metabolic pathways.
Results
Overview of small RNA profiles
To characterize the small RNAs that are responsive to C, N, or S deficiency, 10-day-old seedlings grown on full nutrient (FN), sucrose-free (–C), nitrogen-free (–N), or sulfate-free (–S) MS medium were used to construct small RNA libraries. Compared with the FN seedlings, nutrient-deficient seedlings displayed significant nutrient starvation symptoms. –C seedlings produced short primary roots; –N seedlings displayed significantly delayed leaf growth; and –S seedlings gave produced long lateral roots (Fig. 1A).
By deep sequencing on the Solexa platform, 9,681,350; 10,860,761; 10,285,363; and 9,810,707 reads were generated from FN, –C, –N and –S libraries, respectively. After removing adaptor sequences and low-quality reads, the numbers of sequence reads and unique sequence from the raw data were calculated and then mapped to the Arabidopsis thaliana genome. More than 85% of total sequences were perfectly mapped to the genome, which were further clustered into 11 classes, including exon_antisense, exon_sense, intron_sense, intron_antisense, miRNA, rRNA, repeat, snRNA, snoRNA, tRNA and unannotation (Table 1).
The mapped sequences with lengths ranging from 17 to 25 nucleotides were used to examine the correlation between the length of small RNAs and the proportion of total sequence reads. The most abundant size of small RNAs in the FN library was 21 nucleotides (29.56%), followed by 24 nucleotides (23.67%). The small RNA size distribution pattern in –N was similar to those in FN. In contrast, the small RNAs in –C and –S displayed a different distribution pattern; 20 and 24-nucleotide-length small RNAs were the most abundant in –C and –S, respectively (Fig. 1B). These results suggested that different nutrient supplies caused differential distribution patterns of the sizes of small RNAs.
Profile of known miRNAs
To calculate the number of known miRNAs in each library, we aligned all the unique sequences with lengths between 19 and 26 to all annotated miRNA precursor sequences in Arabidopsis (miRbase Release 18.0, www.miRbase.org). In total, 3,909,038; 5,344,206; 3,968,586; and 3,378,379 mapped sequences were identified for each library, respectively. To compare the miRNA abundance in the different libraries, the abundance of each miRNA in a library was normalized to transcripts per million (TPM) to represent the relative read frequency in each library. miRNAs with an abundance <5 TPM in each library were excluded. The remaining 133 unique mature miRNAs belonged to 60 miRNA families (Supplemental Table 1). In all four libraries, the miR156 family was the most abundant, followed by the miR167 and miR166 families. Out of the 60 miRNA families, 28 families have more than two distinct miRNA sequences. Unlike northern blotting, deep sequencing can be used to analyze different miRNA species from the same family. Based on the sequencing results, the major differences in abundance for different miRNA members from the same family were calculated, such as for the miR156, miR158 and miR159 families. Differential responses of miRNA members to the same nutrient deficiency were also revealed. For example, miR164a and miR164b were downregulated 14-fold by –C, whereas miR164c was not responsive to –C. Similar cases also occurred in the miR169 family and others. These observations implied that different members from the same family might play distinct functions in response to nutrient deficiencies.
miRNAs responsive to a specific nutrient deficiency
To explore the miRNAs that were differentially expressed in response to a specific nutrient deficiency, we compared the read counts of miRNAs under nutrient-deficient conditions with those under FN. The miRNAs with greater than 1.5-fold relative change in sequence counts were identified as differentially expressed miRNAs (Fig. 2). Ninety-two, 79 and 59 differentially expressed miRNAs, which are clustered into 40, 41 and 31 miRNA families, were obtained for –C, –N and –S, respectively (Supplemental Table 1).
miR163, miR169b/c, miR170, miR391, miR447, miR843 and miR848 were specifically upregulated, whereas miR159, miR162, miR164a/b, miR165, miR169d–g, miR172c/d, miR173, miR319, miR773 and miR864-3p were specifically downregulated by –C conditions. Under –N conditions, miR165, miR167c, miR171b/c, miR172c–e, miR773, miR823, miR824, miR826, miR829.1 and miR842 were induced specifically, whereas miR157d, miR158a, miR161.2, miR400, miR447, miR822, miR833-5p, miR843 and miR852 were suppressed. In contrast, fewer differentially expressed miRNAs were identified under –S conditions: miR164c and miR395 were upregulated specifically and miR391 and miR845a were downregulated. Although there have been no reports about –C-responsive miRNAs, previous studies on –N and –S-responsive miRNAs showed that miR169 and miR395 are negatively and positively responsive to –N and –S, respectively21,25. Given that miR169 members displayed differential responses to –N, we calculated all miR169 mature sequence counts, which revealed that the total number of miR169 mature sequences in –N was two-fold less than that in FN (Supplemental Table 2). miR395 was induced sharply by –S. Both downregulation of miR169 by –N and upregulation of miR395 by –S were consistent with previous reports, confirming the reliability of the sequencing data.
To investigate the putative functions of these specifically nutrient-responsive miRNAs, we analyzed their target genes (Supplemental Table 4). Most of the targets are involved in development, stress responses and nutrient metabolism, which are closely linked with nutrient supply conditions.
miRNAs commonly responsive to –C, –N and –S
To identify the miRNAs that are commonly responsive to all three nutrient deficiencies, we compared differentially expressed miRNAs among –C, –N and –S: 16 miRNA families were identified (Fig. 2 and Table 2). The responses of these miRNAs to various nutrient deficiencies were further confirmed by real-time PCR (Supplemental Table 3). miRNA members from 11 miRNA families were downregulated, whereas one family was upregulated, by –C, –N and –S. Among the 11 downregulated miRNA families, eight are directly associated with metabolic process. For example, miR398, miR397, miR408 and miR857 target Superoxide Dismutase (CSD1, CSD2 and CCS1), Cytochrome oxidase c, Laccase (LAC2/3/4/7/12/13/17) and Plantacyanin to regulate the copper starvation (–Cu) response and homeostasis24,33. miR399, miR827 and miR2111 play key roles in the phosphate starvation response and homeostasis. miR399 targets PHO2 which encodes a ubiquitin E2 conjugase and regulates the allocation of phosphate22,34. The target of miR827 is NLA (Nitrogen Limitation Adaptation), the product of which mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis35. miR2111 is specifically induce by P limitation and its target gene is a Kelch repeat-containing F-box gene with unknown function28. miR775 functions in secondary metabolite biosynthesis by targeting a gene that encodes a galactosyltransferase36. In contrast, among the remaining three repressed miRNA families, miR167 and miR172 participate in the auxin response37 and the juvenile-to-adult transition38, respectively and the function of miR841 is unknown. The only miRNA family induced commonly by –C, –N and –S was miR160, which is involved in the auxin response by targeting ARF10, ARF16 and ARF1739,40.
In contrast to the miRNAs that were regulated similarly by –C, –N and –S, miR169b/c, miR395, miR822 and miR837-3p were regulated differentially by –C, –N and –S. miR169b/c was induced by –C, but suppressed by –N and –S. miR395 was positively regulated by –S, but negatively by –C and –N. miR826 was upregulated by –N, but downregulated by –C and –S. miR837-3p decreased in response to –C, but increased to –N and –S. Interestingly, the three miRNA families (miR399, miR827 and miR2111) induced by phosphate starvation (–P) were also repressed by other nutrient deficiencies (–C, –N and –S). This result implied that miRNAs induced specifically by one nutrient deficiency are suppressed by other nutrient deficiencies. To confirm whether this case also occurs for other miRNAs that are induced by other specific nutrient deficiency, we retrieved and analyzed the small RNA sequencing data generated under –P conditions28. Although the miR826 sequence was not found because of low expression abundance in –P, we found that –C-induced miR169b/c, –S-induced miR395 and –Cu-induced miRNAs (miR397, miR398, miR408 and miR857) were significantly suppressed in roots grown in –P conditions (Supplemental Table 5). The interaction between different nutrient metabolisms is common and involves a large number of genes in plants. Our results indicated that many miRNA families that directly participate in the regulation of certain specific nutrient metabolism processes were differentially expressed in response to different nutrient deficiencies. Therefore, it is likely that nutrient-responsive miRNAs also mediate the crosstalk between different nutrient metabolism processes.
Expression correlation between miRNAs and their targets
Plant miRNAs mediate target mRNA cleavage or translation inhibition; therefore, the expression of plant miRNAs is usually negatively correlated with that of their targets. To test this, the expression patterns of miRNAs and their targets were compared using quantitative RT-PCR assays. As expected, most targets displayed the opposite expression trends compared with the corresponding miRNAs. For example, miR169b/c, miR826 and miR395a/d/e were induced by –C, –N and –S, respectively and their targets were repressed correspondingly, with the exception of APS3 (Fig. 3A–C). The expression patterns of miR826-AOP2 in –N and miR395-APS1/3/4 in –S agreed with the previous reports21,26,41. miR397 and miR408 are involved in the copper starvation response. Consistent with the sequencing data, their expression was downregulated by –C, –N and –S. Among the six target genes examined, the expressions of LAC4 and LAC17 were inversely related to miR397b (Fig. 3D), as were those of LAC3 and LAC13 to miR408 (Fig. 3G). Both miR399 and miR827 were suppressed by –C, –N and –S and their targets, PHO2 and NLA, were induced correspondingly (Fig. 3E,H). miR160 and miR167 are involved in the auxin pathway by targeting ARF genes. miR160 was induced whereas miR167a/b was repressed by –C, –N and –S. In contrast, the targets of miR160, ARF10 and ARF16, were repressed, whereas the target of miR167, ARF8, was induced by –C, –N and –S (Fig. 3F,I). In addition, some miRNAs were positively correlated with their targets, such as miR395-APS3 in –S, miR397-LAC2 in –C and miR160-ARF17 in –C and –S. It is likely that these targets are also regulated by other transcription factors or their expression does not completely overlap with miRNAs spatiotemporally.
Altered miRNA expression caused differential adaptation to nutrient deficiency
Overexpression of a certain nutrient-responsive miRNA altered the plant’s adaptation to nutrient starvation conditions21,22,25,27. To investigate the functions of nutrient-responsive miRNAs in nutrient starvation adaptation, miRNA overexpression plants (miR160a-ox, miR395a-ox and miR399b-ox) and miR160 suppression plants (STTM160) were used to evaluate phenotypes in nutrient starvation conditions. As shown in Fig. 4A, miR160a-ox, STTM160 and miR399b-ox plants produced short primary roots compared with wild-type plants under –C conditions. Under –N conditions, miR399b-ox plants developed lateral roots, whereas the wild-type and the other transgenic plants had no visible lateral roots. Under –S conditions, the lateral roots of miR160-ox and miR395a plants were longer than the other plants. The shoot:root ratio is one measure to assess the growth status of plants. The shoot:root ratio of miR160a-ox plants was lower under –C and –S conditions, but higher under –N conditions compared with the other plants. STTM160 and miR399b plants had lower shoot:root ratios than the other plants under –C and –S conditions. In contrast, miR395a-ox plants only displayed a differential shoot:root ratio under –S conditions. These results suggested that misexpression of nutrient-responsive miRNAs leads to altered adaptation to nutrient deficiency.
Discussion
Specificity of miRNA regulation under nutrient-deficient conditions
Although a large number of miRNAs were differentially expressed in response to nutrient deficiencies, some miRNAs were specifically responsive to specific nutrient depletions. miR169b/c, miR826 and miR395 showed the largest changes in response to –C, –N and –S, respectively. Our previous studies confirmed that miR826 regulates the N starvation adaptation of Arabidopsis by reducing glucosinolate synthesis26 and miR395 mediates S homeostasis by regulating sulfate assimilation and transport21. Despite the implication of miR169 in N starvation response, miR169b/c was induced specifically by –C, implying that miR169b/c plays a particular role in C starvation response. Further discussion on miR169 is provided below. Recently, miR156, which mediates juvenile-to-adult phase transition, was confirmed to be suppressed by the addition of sucrose42. Correspondingly, we found that miR156 (except for miR156 h) was specifically induced by the depletion of sucrose. It implies that C status directly affects the abundance of miR156.
Notably, two negative regulators of leaf senescence, miR164a/b and miR319, were specifically repressed by –C. Leaf senescence allows for the degradation of the nutrients produced during the growth phase of the leaf and their reallocation to developing tissues or organs to maximize the fitness of the plant43. miR164, whose expression gradually decreased with leaf aging, targets NAC2/ORE1, which functions positively in leaf senescence44. The miR319-regulated clade of TCP transcription factor genes facilitates the biosynthesis of the hormone jasmonic acid, which then accelerates leaf senescence45. It is likely that carbon starvation induces leaf senescence by suppressing the expression of miR164 and miR319. Although leaf senescence is a common symptom induced by nutrient deficiencies, the expression of both miRNAs was not responsive to –N and –S. This suggested that leaf senescence regulation mediated by miR164 and miR319 is specific to –C, whereas –N and –S induce leaf senescence by other regulatory pathways.
C and N metabolites can function as signals to influence many cellular processes by regulating gene expression in plants. C and N metabolites participate in developmental processes, including flowering time46, root architecture modulation47 and several metabolic pathways (e.g., N assimilation and amino acid synthesis)48,49. A previous study confirmed that a complex C/N-responsive gene network exists in plants and the balance between C and N affects global gene expression7. Under –C conditions, miR447 was induced, whereas –N conditions suppressed miR447. The direct target of miR447 encodes a p-loop containing nucleoside triphosphate hydrolase, which is involved in secondary metabolite biosynthesis50. This result implied that the C-N balance mediates regulation of secondary metabolite biosynthesis by affecting the expression of miR447. In addition, the responses of miR165, miR172c/d/e and miR773 to C starvation were opposite to that to N starvation. Under N limitation conditions, miR165, miR172c/d/e and miR773 were upregulated whereas under C starvation conditions, they were downregulated. miR165 affects shoot apical meristem development via the regulation of HD-ZIP III genes51,52; miR172 mediates juvenile-to-adult transition by the suppression of several AP2 transcription factors53; and miR773 targets a gene that encodes a DNA methyltransferase36. Therefore, it is likely that these three miRNAs mediate the growth and development of plants in response to the C and N starvation.
In addition to conserved miRNAs, some Arabidopsis-specific miRNAs were also involved in the response to nutrient deficiencies (Supplemental Table 4). For example, miR163, miR773, miR843, miR848 and miR854-3p were responsive to –C; miR158a, miR161.2, miR400, miR447, miR773, miR822, miR823, miR826, miR833-5p, miR843 and miR852 were responsive to –N; and miR845a was responsive to –S. Although the targets of most Arabidopsis-specific miRNAs are unknown, the target genes of miR158, miR163 and miR826 encode glycosyltransferase, methyltransferases and 2-oxoglutarate-dependent dioxygenase, all of which play roles in secondary metabolite biosynthesis26,41,54. This result implied that nutrient deficiencies affect the biosynthesis of secondary metabolites indirectly by altering abundance of miRNAs.
Universality of miRNA regulation under nutrient-deficient conditions
miR169 was repressed by –N and regulated nitrogen homeostasis in Arabidopsis25. Interestingly, the total number of miR169 mature sequences was also downregulated by –S (Supplemental Table 2). A similar repression of miR169 was observed under –P conditions in Arabidopsis28. These observations suggested that miR169 might not only have a specific function in nitrogen homeostasis, but also in S and P metabolism. Notably, different miR169 members showed differential responses to –C, –N and –S, as well as to –P (Supplemental Table 2 and 5). With other miR169 species unaffected or reduced, miR169b/c was dramatically induced by –C. A previous study revealed that –N specifically upregulated miR169d–g, but not other miR169 species21. In contrast, –S and –P suppressed nearly all miR169 species. Therefore, it is likely that miR169b/c and miR169d–g specifically affect C and N metabolism, respectively. There are one or two nucleotide differences between miR169 species, implying that different miR169 mature sequences may have different target genes, as described in a recent study, which confirmed that miR157d, but not other species of the miR156/157 family, mediated the cleavage of the HY5 mRNA55.
Nutrient availability is closely linked with root development. Under C or N-free conditions, the root growth was inhibited (Fig. 1A). However, relative to the shoot growth, the root growth displayed significant advantage under –N conditions whereas an opposite case was observed under –C conditions (Fig. 4B). We observed that –S conditions promote root branching (Fig. 1A) although an opposite observation was reported56. The analysis of shoot/root mass ratio suggested that –S relatively promotes root growth compared with the shoot growth (Fig. 4B). It has been revealed that plants employ miRNAs to alter the root system to adapt to the fluctuation of nutrient availability57. Nitrogen treatment led to a reduction in miR167 and the elevation of its target, ARF8, which mediates the balance between lateral root initiation and emergence58. miR160 controls root cap formation, lateral root number and primary root length by mediating the cleavage of its targets, ARF10 and ARF1640. Both sequencing data and quantitative RT-PCR results indicated that miR160 was induced, whereas miR167 was suppressed, by –C, –N and –S, implying that under nutrient deficiency conditions, or at least under –C, –N and –S conditions, plants regulate root development-associated miRNAs to modulate their root systems. As expected, ectopic or disrupted expression of miR160 altered the root systems under both nutrient sufficiency and deficiency conditions (Fig. 4A). The analysis of relative root growth revealed that miR160 overexpression facilitated root growth under –C and –S conditions (Fig. 4B). miR395 plays key roles in modulating sulfate uptake and allocation by targeting genes encoding sulfate assimilation and transport proteins respectively21. Under –S conditions, elevation of miR395 resulted in advantageous root growth. In contrast, overexpression of miR399b inhibited primary root growth, but promoted later root initiation, under –N conditions. These results suggests that nutrient responsive miRNAs can regulate the root system of plants, which likely then affects uptake of nutrient elements.
To maintain normal growth and development, plants must acquire sufficient nutrients and keep the balance of nutrient elements, which are then incorporated into a variety of important compounds with structural and physiological roles. However, the different nutrient metabolic pathways are inter-dependent59. Deficiency of C induced miR169b/c, which was repressed by other nutrient deficiencies, including –N, –S and –P. Similarly, –S upregulated miR395, which was downregulated by –C, –N and –P. This nutrient-responsive pattern appears to be universal to miRNAs that are induced by a specific nutrient deficiency. For example, –Cu induced miRNAs (miR397, miR398, miR408 and miR857) that were suppressed by –C, –N, –S and –P. miR399, miR827 and miR2111 were upregulated by –P, but downregulated by –C, –N and –S. It implies that miRNAs that are induced specifically by a certain nutrient deficiency are often suppressed by other nutrient deficiencies and that this likely prevents the imbalance of different nutrient elements in the plants because the downregulation of miRNAs can cause reduced uptake of the corresponding miRNA-associated nutrient elements. When miR395a-ox and miR399b-ox plants were subject to nutrient starvation treatments, they displayed significant morphological differences and altered adaptations to nutrient deficiencies, compared with wild-type plants (Fig. 4). Therefore, these nutrient deficiency-induced miRNAs may function as signals that mediate the crosstalk between nutrient metabolic pathways. Thus, it can be hypothesized that plants have developed a response to avoid imbalances in nutrient supply by modulating a suite of miRNAs that function in specific nutrient metabolic pathways.
Methods
Plant growth conditions
Arabidopsis thaliana (ecotype Columbia) seeds were used in this study. Sterilized seeds were suspended in 0.1% agarose and plated on MS medium. Plates were put in darkness for 2 d at 4 °C and then transferred to a tissue culture room at 22 °C under a 16-h-light/8-h-dark photoperiod.
For nutrient starvation experiments, plants were grown in long-day conditions on modified MS/agar media, containing 0.8% Agar A. For full nutrient, MS medium was used. For –C, the MS medium without the addition of sucrose was used. For –N, MS medium in which ammonium nitrate was removed and potassium nitrate was replaced with equivalent amounts of potassium chloride was used. For –S, the sulfate-containing salts of the MS medium were replaced with equivalent amounts of chloride salts. Root and shoot samples for RNA isolation were collected from 10-day-old seedlings grown vertically on MS medium.
RNA preparation and small RNA sequencing
The Trizol reagent (Invitrogen) was used to extract total RNA. PEG8000/NaCl precipitation was used to isolate low molecular weight RNA from 200 μg total mRNAs. Small RNAs in the size range of 20 to 30 nucleotides were purified from 15% denaturing polyacrylamide gels and ligated first with the 5′ RNA adaptor and then with the 3′ RNA adaptor. At each step, the ligated products were purified by electrophoretic separation on polyacrylamide gels. After first-strand synthesis and 18 cycles of PCR amplification, the final bands were purified on PAGE gels and submitted for sequencing. The Beijing Genomics Institute (BGI) performed the sequencing.
Computational analysis of sequencing data
The raw sequencing data were trimmed by removing adaptor sequences and mapped to the Arabidopsis genome (The Arabidopsis Information Resource release version 10; http://www.arabidopsis.org/). Reads perfectly matching those in the Arabidopsis genome were used for further analysis. Arabidopsis mature miRNAs and their precursors were retrieved from miRBase (http://www.mirbase.org).
Construction of transgenic plants
The construction of miR160a-ox and miR395a-ox transgenic plants was reported previously21,41. For the construction of miR399b transgenic plants, a construct in which the CaMV35 promoter drove the expression of the DNA sequence containing the putative miR399b precursor was introduced into wild-type plants. For construction of STTM160 transgenic plants, a short tandem target mimic for miR160 was designed according to a previous report60.
Gene expression analysis
Seedling samples were harvested separately from Arabidopsis and frozen in liquid nitrogen for storage at −80 °C. The Trizol reagent (Invitrogen) was used to isolate total RNA, which was digested by DNaseI (Fermentas). Stem-loop RT-PCR was used to detect the expression of miRNAs. To produce miRNA-fused stem-loop cDNA, 0.5 μg total RNA was used for reverse transcription with miRNA mature-sequence-specific stem-loop RT primers, according to the stem-loop RT-PCR protocol61. For mRNA cDNA, 1 μg of total RNA was reverse-transcribed using an oligo(dT)18 primer, according to the reverse transcription protocol (Takara). A 20-μl reaction mixture was used for the production of cDNA. After heat inactivation, a 1-μl aliquot was used as the template for real-time quantitative RT-PCR. An miRNA-specific primer and a universal primer were used to amplify miRNA-fused cDNA. Two specific primers were used to amplify each miRNA target gene. All primers used in this study are listed in Supplemental Table 6. Arabidopsis ACT2 (At3g18780) was used as an internal control for real-time RT-PCR. A SYBR Premix Ex TaqTM kit (TaKaRa) on a Roche LightCycler 480 real-time PCR machine performed all the quantitative RT-PCR analyses, according to the manufacturer’s instructions.
Additional Information
How to cite this article: Liang, G. et al. Uncovering miRNAs involved in crosstalk between nutrient deficiencies in Arabidopsis. Sci. Rep. 5, 11813; doi: 10.1038/srep11813 (2015).
Change history
20 April 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
References
Zheng, Z. Carbon and nitrogen nutrient balance signaling in plants. Plant Signaling & Behavior 4, 584–591 (2009).
Krapp, A. et al. Arabidopsis roots and shoots show distinct temporal adaptation patterns toward nitrogen starvation. Plant Physiol 157, 1255–1282 (2011).
Wang, R., Okamoto, M., Xing, X. & Crawford, N. M. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron and sulfate metabolism. Plant Physiol 132, 556–567 (2003).
Scheible, W. R. et al. Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol 136, 2483–2499 (2004).
Palenchar, P. M., Kouranov, A., Lejay, L. V. & Coruzzi, G. M. Genome-wide patterns of carbon and nitrogen regulation of gene expression validate the combined carbon and nitrogen (CN)-signaling hypothesis in plants. Genome Biol 5, 91 (2004).
Price, J., Laxmi, A. St, Martin, S. K., & Jang, J. C. Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis. Plant Cell 16, 2128–2150 (2004).
Gutiérrez, R. A. et al. Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis. Genome Biol 8, R7 (2007).
Kopriva, S. et al. Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana. Plant J 20, 37–44 (1999).
Kopriva, S. et al. Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor. Plant Physiol 130, 1406–1413 (2002).
Hesse, H. et al. Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots. J Exp Bot 54, 1701–1709 (2003).
Yoshimoto, N., Inoue, E., Watanabe-Takahashi, A., Saito, K. & Takahashi, H. Posttranscriptional regulation of high-affinity sulfate transporters in Arabidopsis by sulfur nutrition. Plant Physiol 145, 378–388 (2007).
Yoshimoto, N., Takahashi, H., Smith, F. W., Yamaya, T. & Saito, K. Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots. Plant J 29, 465–473 (2002).
Maruyama-Nakashita, A., Nakamura, Y., Yamaya, T. & Takahashi, H. Regulation of high-affinity sulphate transporters in plants: towards systematic analysis of sulphur signalling and regulation. J Exp Bot 55, 1843–1849 (2004).
Prosser, I., Purves, J., Saker, L. & Clarkson, D. Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate. J Exp Bot 52, 113–121 (2001).
Radin, J. W. & Eidenbock, M. P. Carbon Accumulation during Photosynthesis in Leaves of Nitrogen- and Phosphorus-Stressed Cotton. Plant Physiol 82, 869–871 (1986).
Schachtman, D. P. & Shin, R. Nutrient sensing and signaling: NPKS. Annu Rev Plant Biol 58, 47–69 (2007).
Gojon, A., Nacry, P. & Davidian, J. C. Root uptake regulation: a central process for NPS homeostasis in plants. Curr Opin Plant Biol 12, 328–338 (2009).
Hafsi, C., Debez, A. & Abdelly, C. Potassium deficiency in plants: effects and signaling cascades. Acta Physiol Planta 36, 1055–1070 (2014).
Ye, Y. et al. Carbon, Nitrogen and Phosphorus Accumulation and Partitioning and C:N:P Stoichiometry in Late-Season Rice under Different Water and Nitrogen Managements. PLoS One 10.1371/journal.pone.0101776 (2014).
Voinnet, O. Origin, Biogenesis and Activity of Plant MicroRNAs. Cell 136, 669–687 (2009).
Liang, G., Yang, F. X. & Yu, D. Q. MicroRNA395 mediates regulation of sulfate accumulation and allocation in Arabidopsis thaliana. Plant J 62, 1046–1057 (2010).
Chiou, T. J. et al. Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell 18, 412–421 (2006).
Sunkar, R., Kapoor, A. & Zhu, J. K. Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18, 2051–2065 (2006).
Abdel-Ghany, S. E. & Pilon, M. MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 283, 15932–15945 (2008).
Zhao, M., Ding, H., Zhu, J. K., Zhang, F. & Li,W. X. Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis. New Phyt 190, 906–915 (2011).
He, H., Liang, G., Li, Y., Wang, F. & Yu, D. Two young MicroRNAs originating from target duplication mediate nitrogen starvation adaptation via regulation of glucosinolate synthesis in Arabidopsis thaliana. Plant Physiol 164, 853–865 (2014).
Kant, S., Peng, M. & Rothstein, S. J. Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. Plos Genet 7, e1002021 (2011).
Hsieh, L. C. et al. Uncovering small RNA-Mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151, 2120–2132 (2009).
Pant, B. D. et al. Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol 150, 1541–1555 (2009).
Zhang, S., Sun, L. & Kragler, F. The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation. Plant Physiol 150, 378–387 (2009).
Buhtz, A., Pieritz, J., Springer, F. & Kehr, J. Phloem small RNAs, nutrient stress responses and systemic mobility. BMC Plant Biol 10, 64 (2010).
Pant. B. D., Buhtz, A., Kehr, J. & Scheible, W. R. MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53, 731–738 (2008).
Beauclair, L., Yu, A. & Bouché N. microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis. Plant J 62, 454–462 (2010).
Lin, W. Y., Huang, T. K., Leong, S. J. & Chiou, T. J. Long-distance call from phosphate: systemic regulation of phosphate starvation responses. J Exp Bot 65, 1817–1827 (2014).
Lin, W. Y., Huang, T. K. & Chiou, T. J. Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. Plant Cell 25, 4061–4074 (2013).
Fahlgren, N. et al. High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes. PLoS ONE 2, e219 (2007).
Wu, M. F., Tian, Q. & Reed, J. W. Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression and regulates both female and male reproduction. Development 133, 4211–4218 (2006).
Poethig, R.S. Vegetative phase change and shoot maturation in plants. Curr Top Dev Bio 105, 125–152 (2013).
Mallory, A., Bartel, D. P. & Bartel, B. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360–1375 (2005).
Wang, J. W. et al. Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17, 2204–2216 (2005).
Liang, G., He, H., & Yu, D. Identification of nitrogen starvation-responsive microRNAs in Arabidopsis thaliana. PLoS One 7, e48951 (2012).
Yu, S. et al. Sugar is an endogenous cue for juvenile-to-adult phase transition in plants. Elife. 10.7554/eLife.00269. (2013).
Woo, H. R., Kim, H. J., Nam, H. G. & Lim, P. O. Plant leaf senescence and death regulation by multiple layers of control and implications for aging in general. J Cell Sci 126, 4823–4833 (2013).
Kim, J. H. et al. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323, 1053–1057 (2009).
Schommer, C. et al. Control of jasmonate biosynthesis and senescence by miR319 targets. PLoS Biol 6, e230 (2008).
Corbesier, L., Lejeune, P. & Bernier, G. The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant. Planta 206, 131–137 (1998).
Lynch, J. Root architecture and plant productivity. Plant Physiol 109, 7–13 (1995).
Koch, K. E. Carbohydrate-modulated gene expression in plants. Ann Rev Plant Physiol Mol Bio 47, 509–540 (1996).
Stitt, M. Nitrate regulation of metabolism and growth. Curr Opi Plant Biol 2, 178–186 (1999).
Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005).
Zhou, G. K., Kubo, M., Zhong, R., Demura, T. & Ye, Z. H. Overexpression of miR165 affects apical meristem formation, organ polarity establishment and vascular development in Arabidopsis. Plant Cell Physiol 48, 391–404 (2011).
Zhu, H. et al. Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242–256 (2011).
Wu, G. et al. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138, 750–759 (2009).
Ng, D. W. et al. cis- and trans-Regulation of miR163 and target genes confers natural variation of secondary metabolites in two Arabidopsis species and their allopolyploids. Plant Cell 23, 1729–1740 (2011).
Tsai, H. et al. HUA ENHANCER1 is involved in posttranscriptional regulation of positive and negative regulators in Arabidopsis photomorphogenesis. Plant Cell 10.1105/tpc.114.126722 (2014).
Dan, H., Yang, G. & Zheng, Z. A negative regulatory role for auxin in sulphate deficiency response in Arabidopsis thaliana. Plant Mol Biol 63, 221–235 (2007).
Meng, Y., Ma, X., Chen, D., Wu, P. & Chen, M. MicroRNA-mediated signaling involved in plant root development. Biochem Bioph Res Co 393, 345–349 (2010).
Gifford, M. L., Dean, A., Gutieerez, R. A., Coruzzi, G. M. & Birnbaum, K. D. Cell-specific nitrogen responses mediate developmental plasticity. P Natl Acad Sci USA 105, 803–808 (2008).
Ohkama-Ohtsu, N. & Wasaki, J. Recent progress in plant nutrition research: cross-talk between nutrients, plant physiology and soil microorganisms. Plant Cell Physiol 51, 1255–1264 (2010).
Yan, J. et al. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415–427 (2012).
Varkonyi-Gasi, E., Wu, R., Wood, M., Walton, E. F. & Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Meth 3, 12 (2007).
Acknowledgements
We are grateful for the support of the National Natural Science Foundation of China [Grant No. 31100186] and the West Light Foundation of CAS.
Author information
Authors and Affiliations
Contributions
G.L. designed the research; G.L. and Q.A. performed experiments; G.L., Q.A. and D.Q.Y analyzed data; G.L. prepared the figures and tables; G.L. wrote the manuscript; All authors reviewed the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Liang, G., Ai, Q. & Yu, D. Uncovering miRNAs involved in crosstalk between nutrient deficiencies in Arabidopsis. Sci Rep 5, 11813 (2015). https://doi.org/10.1038/srep11813
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep11813
This article is cited by
-
MicroRNAs modulating nutrient homeostasis: a sustainable approach for developing biofortified crops
Protoplasma (2023)
-
MicroRNA775 Targets a Probable β-(1,3)-Galactosyltransferase to Regulate Growth and Development in Arabidopsis thaliana
Journal of Plant Growth Regulation (2022)
-
MiRNA fine tuning for crop improvement: using advance computational models and biotechnological tools
Molecular Biology Reports (2022)
-
Micro RNA mediated regulation of nutrient response in plants: the case of nitrogen
Plant Physiology Reports (2022)
-
Downregulation of GeBP-like α factor by MiR827 suggests their involvement in senescence and phosphate homeostasis
BMC Biology (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.