ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № б, с. 844-849
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
MicroRNA399 EXPRESSION PROFILES IN Arabidopsis SEEDLINGS, CALLUS, AND PROTOPLASTS IN RESPONSE TO PHOSPHATE DEFICIENCY1
© 2014 P. Zhao* 2, F. Liu** 2, H. Liu**
*Key Laboratory of Stress Physiology and Ecology in Cold and Arid Regions, Gansu Province, Cold and Arid Regions Environmental and Engineering Research Institute, CAS, Lanzhou,
P.R. China
**Institute of Cell Biology, Life Science School, Lanzhou University, Lanzhou, P.R. China
Received November 27, 2013
The functions of microRNA399 (miR399) in response to phosphate (Pi) deficiency have been extensively studied in Arabidopsis; however, previous studies have focused on relatively late responses of seedlings. In this study, the expression profiles of five miR399 primary transcripts (pri-miR399s) and mature miR399 were investigated in seedlings, calli, and mesophyll protoplasts. Pi deficiency rapidly stimulated the accumulation of pri-miR399s in the seedlings except pri-miR399b at 1 day; the amount of pri-miR399a decreased at 3 and 5 days. pri-miR399c and pri-miR399e/f showed continuously increasing patterns; the greatest accumulation of pri-miR399d was observed at 3 days. The expression of pri-miR399b, c, and f was significantly induced in the callus after 15 days of exposure to Pi deficiency. In protoplasts, the expression patterns of five pri-miR399s were comparable to those in seedlings at 1 day of Pi deficiency. Mature miR399 accumulated in the treated seedlings; and more than 460-fold of induction was observed in the calli without Pi. The expression profiles of pri-miR399s suggest that these primary transcripts are temporally and tissue-specific regulated in plant responses to Pi deficiency. Moreover, fresh isolated protoplasts could be used to study physiological perception and local signaling of Pi deficiency during early stages.
Keywords: Arabidopsis — microRNA399 — phosphate deficiency — expression profile — callus — protoplast
DOI: 10.7868/S0015330314060232
INTRODUCTION
Phosphate (Pj) availability is a limiting factor of plant growth, development, and yield [1, 2]. To cope with Pj deficiency, plants have developed sophisticated and tightly controlled mechanisms and thus maintain Pj homeostasis in acquisition, storage, allocation, and recycling of plants [1, 3, 4]. Various adaptive strategies are involved in these processes, such as reprogramming their transcriptome, proteome, and metabolome. Recent advances have unveiled that several important components, including transcription factors, SPX domain-containing proteins, and proteins involved in posttranslational modification processes, such as phosphorylation, dephosphorylation, and small
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations'. CIM — callus induction medium; IPS1 — induced by Pi starvation 1; miRNA — micro RNA; PHR1 — phosphate starvation response 1; pri-miR399s — primary transcripts of miR399s.
Corresponding author. Heng Liu. Institute of Cell Biology, Life Science School, Lanzhou University, 222# Tianshui Nan Lu, Lanzhou 730000, P.R. China; e-mail. hengliu@lzu.edu.cn
ubiquitin-like modifier conjugation (SUMOylation), are involved in a coordinated network that adapts plants to Pj limitation [1—5]. Moreover, sugars and phytohormones, such as gibberellins, ethylene, auxins, cytokinins, abscisic acid, and strigolac tones, as well as ions, such as iron, have crucial functions in mediating gene expression profiles and in altering the root system architecture in response to Pj deficiency [1—3]. Aside from these components, microRNAs (miRNAs), such as miR156, miR169, miR395, miR398, miR399, miR778, miR827, and miR2111, and non-coding RNAs are uncovered as essential for the plant strategy to cope with Pi status [1, 3, 6, 7].
The miR399 is one of the most characterized miRNAs in the regulation of Pi homeostasis in plants [6, 8—11]. Grafting assays have verified that mature miR399 can be transported via phloem from the shoot to the root, where it directs the cleavage of the transcripts of an ubiquitin-conjugating E2 enzyme UBC24/PHO2 [12, 13]. The suppression of PHO2 increases the transcript levels of root Pi transporter genes PHT1;8 and PHT1;9, thereby activating Pi uptake [1, 3, 11]. PHO2 regulates Pi transport by ubiquitin-me-diated protein degradation; the appropriate expression
of PHO2 is crucial to maintain Pi homeostasis under Pi deficiency and sufficient conditions [3]. The accumulation of miR399 upon Pi limitation is positively regulated by a MYB transcription factor, called phosphate starvation response 1 (PHR1) [9]. Furthermore, the cleavage activity of miR399 on PHO mRNA is suppressed by one non-coding RNA AT4/Induced by Pt Starvation 1 (IPS1) [14, 15]. Thus, PHO2, miR399, PHR1, and AT4/IPS1 define a confined Prsignaling pathway in Arabidopsis.
Northern blot has played an important role in the validation and quantification of miRNA expression [6, 8—10, 15, 16]. However, this method has some drawbacks, such as poor sensitivity to detect few miRNAs and a complex protocol [17]. With technological advancement [17, 18], quantitative real-time PCR (qPCR) has been performed to monitor the expression ofmature miRNAs successfully [9, 12, 13]. In Arabidopsis, six miR399s, namely, miR399a to miR399f, which are generated from five primary transcripts (pri-miR399s), have been uncovered; miR399e and miR399fare encoded by the same gene [9, 13, 16]. According to the accumulation level of primary transcripts, pri-miR399d is one of the most prominent transcripts under Prdeprived conditions. Time-course monitoring of pri-miR399 expression showed that miR399 specifically responded to Pi deprivation; the high abundance of pri-miR399s in Prdeprived seedlings decreased rapidly at resupplying Pi after 30 min [9]. As a systemic signal, miR399 has a relatively long half-life, approximately 12 h in Arabidopsis [3, 9, 13]. The expression patterns of pri-miR399s and mature miR399 have been extensively studied in Arabidopsis [9, 12, 13]; however, most of these studies were focused on the relative long seedling treatment periods. Studies on pri-miR399s and miR399 in seedlings, calli, and protoplasts of Arabidopsis will further deepen our insight into the function of miR399.
MATERIALS AND METHODS
Plant materials and treatments. Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. Seeds were surface-sterilized, planted on MS medium with 3% sucrose and 1% agar, and synchronized for 3 days at 4°C. The seeds were then placed under long-day (a 16-h photoperiod) conditions for 7 days.
Seedling treatments. For seedling treatments, the seven-day-old seedlings were transferred to normal MS or MS media without Pi for 1, 3, and 5 days. After these different treatments, the seedlings were collected to extract total RNA.
Callus treatments. Leaf explants were excised from the seedlings in 15 days after germination. The leaves were cut into small pieces and transferred to the callus induction medium (CIM): MS medium with 3 mg/L
2,4-D, 0.05 mg/L kinetin, 0.5 g/L MES hydrate, 3% sucrose, and 1% agar. The induced callus was transferred to new CIM (CIM+Pj) or CIM without Pi (CIM—Pj) and incubated for 15 days to extract the total RNA.
Protoplast treatments. Mesophyll protoplasts were isolated from four-week-old rosette leaves according to Yoo et al. [19] and then transferred to CIM or CIM—Pi for 36 h. Both media were supplied with 0.4 M mannitol; agar was removed. After treatments, the protoplasts were sedimented by centrifugation at 100 g. The total RNA was then extracted.
RNA extraction and real-time PCR analysis. Total RNA was isolated from the collected materials with TRIzol ("Invitrogen™, Life Technologies", United States) and then treated with RNase-free DNase I ("TaKaRa", China) according to the manufacturer's instruction. To detect the primary transcripts of miR399, 0.5 ^g of total RNA was reverse transcribed using the RevertAid—TM First Strand cDNA Synthesis Kit ("Fermentas", Canada); 1 ^L of oligodT and 1 ^L of random primers were also included in the 20-^L reaction system. The RT2 primer described by Pant et al. [13] was used to synthesize cDNA to detect miR399d primary transcripts. Quantitative real-time PCR was conducted using the primer pairs described previously [9]. Actin2 (At3g18780) was used as an internal control [20]. According to Chen et al. [18] and Yang et al. [17], 2 ^L of10 ^M miR399 specific stem-loop prime [13] and 0.4 ^L of 100 pM actin reverse primer were added to the cDNA synthesis system. SYBR® Premix Ex TaqTM II ("TaKaRa") Kit was used for real-time monitoring of DNA amplification by Rotor-gene 3000.
RESULTS AND DISCUSSION
Expression profiles of pri-miR399s and mature miR399 in seedlings in response to Pt deficiency
Previous time-course assays demonstrated that the expression of four pri-miR399s (a, c, d, and e) increased significantly in 5 days after Pi deprivation; the highest accumulation was observed in 9 days [9]. Pri-miR399d is the most prominent one among pri-miR399s [9]. Pri-miR399s cannot be expressed in the roots in the first 2 to 3 h upon Pi deficiency treatments [13]. To investigate the early response of seedlings under Pi deficiency conditions, the expression profiles of five pri-miR399s and mature miR399 were analyzed by qPCR at 1, 3, and 5 days during the development of Pi deficiency (fig. 1). The five pri-miR399s were induced by Pi deficiency treatments with different temporal expression patterns. Pri-miR399a, c, d, and e/fwere strongly and rapidly induced after 1 day of exposure to Pi deficiency. Pri-miR399b expression was slightly induced by the treatment (fig. 1b); the amount of pri-miR399a decreased at 3 and 5 days, but this
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