Cross-talk between Phosphate Starvation and Other Environmental Stress Signaling Pathways in Plants.

The maintenance of inorganic phosphate (Pi) homeostasis is essential for plant growth and yield. Plants have evolved strategies to cope with Pi starvation at the transcriptional, post-transcriptional, and post-translational levels, which maximizes its availability. Many transcription factors, miRNAs, and transporters participate in the Pi starvation signaling pathway where their activities are modulated by sugar and phytohormone signaling. Environmental stresses significantly affect the uptake and utilization of nutrients by plants, but their effects on the Pi starvation response remain unclear. Recently, we reported that Pi starvation signaling is affected by abiotic stresses such as salt, abscisic acid, and drought. In this review, we identified transcription factors, such as MYB, WRKY, and zinc finger transcription factors with functions in Pi starvation and other environmental stress signaling. In silico analysis of the promoter regions of Pi starvation-responsive genes, including phosphate transporters, microRNAs, and phosphate starvation-induced genes, suggest that their expression may be regulated by other environmental stresses, such as hormones, drought, cold, heat, and pathogens as well as by Pi starvation. Thus, we suggest the possibility of cross-talk between Pi starvation signaling and other environmental stress signaling pathways.

INTRODUCTION

The availability of inorganic phosphate (Pi) in soil is a crucial determinant of plant growth and development as well as crop productivity (Raghothama, 1999). Plants have evolved morphological, physiological, biochemical, and molecular processes to improve the mobilization, acquisition, and efficient utilization of Pi under deficiency conditions (Poirier and Bucher, 2002; Yuan and Liu, 2008). Reports on the mechanisms that regulate sensing and the response to Pi starvation have identified Pi starvation signaling pathway components and the cross-talk between Pi starvation responses and other plant signaling pathways, including sugars, phytohormones, and photosynthesis (Franco-Zorrilla et al., 2005; Lei et al., 2011a; Rouached et al., 2010; Rubio et al., 2009).

Cross-regulation occurs between Pi starvation and other plant signaling pathways, such as sugars and phytohormones (Rouached et al., 2010; Yuan and Liu, 2008). Pi starvation often causes sugar accumulation in plant tissues; high sugar levels in roots induce root system architecture (RSA) changes under Pi deprivation (Ciereszko et al., 2005; Hammond and White, 2008). Moreover, exogenous sucrose treatment increases the expression levels of Pi transporters and phosphate starvation–induced (PSI ) genes (Karthikeyan et al., 2007; Lejay et al., 2008; Müller et al., 2005). Sugar signaling is also connected with various hormone signaling pathways under Pi starvation (Gibson, 2004). Auxin and ethylene levels are increased by sucrose in the roots, where they are related to RSA developmental changes in Pi starvation responses (Jain et al., 2007; Ma et al., 2003). Pi and abscisic acid (ABA) signaling pathways mediate developmental processes during RSA changes, including increases in the root:shoot ratio and root hair density (Ciereszko and Kleczkowski, 2002; Trull et al., 1997). The cytokinin receptor CRE1/WOL/AHK4 is implicated in the cross-talk between Pi and cytokinin signal transduction pathways by controlling the transcriptional levels of PSI genes (Franco-Zorrilla et al., 2005; Wang et al., 2006). Thus, Pi starvation signaling is strongly linked with numerous plant signaling pathways to maintain appropriate Pi homeostasis in response to changing environmental conditions.

The signaling networks involved with plant responses to Pi starvation are well known, but the cross-talk between Pi starvation and other abiotic stress signaling pathways remains unclear. Recently, however, their cross-talk has been suggested in physiological, phenotypical, and molecular levels. In barley, heat stress affects the expression of PSI genes, which leads to maintenance of Pi homeostasis in plant tissues (Pacak et al., 2016). They suggest that retarded growth and accelerated senescence of barley under heat stress conditions is probably due to disturbances of the macronutrient, including Pi, homeostasis. Comparative root transcriptome analysis using rice cultivars exhibiting contrasting RSA suggests that plants recruit common molecular machinery controlling different regulatory pathways, such as root development, nutrient signaling, biotic- or abiotic-stress responses (Singh et al., 2016). In addition, the RSA formation under Pi starvation conditions are also adjusted by metal stresses, such as arsenate, iron, and aluminum (Dong et al., 2017). In Arabidopsis accessions sensitivities to Pi deficiency are extremely enhanced by arsenate stress (Shukla et al., 2015). Recent reports mention that the AtMYB2 transcription factor, which functions in abiotic stress signaling pathways in Arabidopsis (Abe et al., 2003; Yoo et al., 2005), also acts as a direct transcriptional activator of the miR-NA399f (miR399f ) gene, which plays a crucial role in maintaining Pi homeostasis (Baek et al., 2013). The miR399f also plays numerous roles in modulating plant responses to abiotic stresses, such as salt, ABA, and drought (Baek et al., 2016). The results indicate that cross-talk occurs between Pi starvation signaling and other abiotic stress signaling pathways.

The transcription factors and cis-acting elements of molecular components involved in the signaling cascade have been analyzed to understand plant signaling regulatory mechanisms (Jain et al., 2012; Liu et al., 2015; Yamaguchi-Shinozaki and Shinozaki, 2005), where some transcription factors play multiple roles in responses to different stresses (Briat et al., 2015; Jain et al., 2012). Thus, in silico analysis of cis-acting regulatory elements in the promoters of stress-responsive genes have clarified the molecular and regulatory mechanisms of cross-talk among several stress signaling pathways. In this review, we summarize the transcription factors that participate in both Pi starvation responses and other signaling responses to phytohormones and biotic and abiotic stresses. We believe that the expression of several genes involved in Pi starvation responses may be mediated via different stress signaling cascades according to in silico analysis of the links between Pi starvation and other stress signaling pathways.

TRANSCRIPTION FACTORS THAT CO-REGULATE PI STARVATION AND OTHER STRESS SIGNALING PATHWAYS

MYB Transcription Factors

MYB transcription factors are associated with the signaling networks in various stress responses (Dubos et al., 2010; Franco-Zorrilla et al., 2004). Phosphate starvation response 1 (PHR1) is a representative MYB transcription factor in Pi starvation response (Rubio et al., 2001). PHR1 and PHR1-like (PHL) belong to the MYB-CC class and they directly bind to PHR1-binding site (P1BS; GNATATNC) or P1BS-like (AC/AATATT/CC) elements in the promoter regions of target genes during the Pi starvation stress (Table 1). PHR1 and PHLs regulate the transcription of Pi starvation response target genes, including Pht, PSI, Pi starvation-responsive, and Pi starvation–induced acid phosphatase genes (Nilsson et al., 2007; Sun et al., 2016). PHR1 primarily acts as a transcriptional activator of Pht1, and PHO1 is necessary for Pi uptake by roots under Pi-deficient conditions (Bayle et al., 2011). PHR1 also controls the transcription of genes, such as FERRITIN 1 and galactolipid synthesis genes in responses to Pi, metals, and oxygen deficiency (Bournier et al., 2013; Briat et al., 2015; Klecker et al., 2014).

Table 1

Transcription factors interconnecting Pi starvation and other stress-responsive signaling pathways in Arabidopsis

Type of Factor	Transcription	Name	Locus	Binding Motif	Sequence	Responses	References
MYB Family	MYB-CC (R1-type)	PHR1	At4g28610	P1BS element (P1BS-like element)	GNATATNC (AC/AATATT/CC)	Pi starvation, metals deficiency, oxygen deficiency	Briat et al., 2015; Bustos et al., 2010; Khan et al., 2014; Klecker et al. 2014; Nilsson et al., 2007; Rubio et al. 2001
		PHL1	At5g29000			Pi starvation	Bustos et al., 2010; Sun et al., 2016
		PHL2	At3g24120			Pi starvation	Sun et al., 2016
		PHL3	At4g13640			Pi starvation	Sun et al., 2016
	MYB-CC (R2R3-type)	MYB2	At2g47190	MBS	TAACTG	Pi starvation, cytokinin response, salt/ABA/drought response	Abe et al., 1997; 2003; Baek et al., 2013; Guo and Gan, 2011; Yoo et al., 2005
		MYB62	At1g68320			Pi starvation, GA deficiency	Devaiah et al., 2009
WRKY	Family	WRKY6	At1g62300	W box	TTGACT/C	Pi starvation, pathogen defense, ABA response	Robatzek and Somssich, 2002; Chen et al., 2009; Huang et al., 2016
		WRKY42	At4g04450			Pi starvation	Su et al., 2015
		WRKY45	At3g01970			Pi starvation	Wang et al., 2014c
		WRKY75	At5g13080			Pi starvation, JA/SA response, pathogen defense	Chen et al., 2013; Devaiah et al., 2007a; Schmiesing et al., 2016
ZFP family	Zinc Finger (C2H2-type)	ZAT6	At5g04340	POS9A POS9B and POS9C DRE	(GA)9 repeat TGTGAGAGATGGCCGAC	Pi starvation, metals stress, salt/drought/osmotic stress response	Chen et al., 2016; Devaiah et al., 2007b; Liu et al., 2013; Nakashima and Yamaguchi-Shinozaki, 2006

MYB2 functions as a transcriptional activator of ABA-dependent or ABA-independent genes under abiotic stress. MYB2 increases the transcriptional level of RD22 by activating its promoter under drought and ABA stress conditions (Abe et al., 1997; Hoeren et al., 1998). Results of microarray analyses using transgenic plants that overexpressed MYC2/MYB2 showed upregulation of RD22, ADH1, COR6.6, and RD20 genes and the presence of MYB-binding sequences in their promoter regions (Abe et al., 2003). MYB2 also activates the transcription of miR399f in the Pi starvation response by directly binding to a MYB-binding site (MBS; TAACTG) motif in the miR399f promoter region (Table 1; Baek et al., 2013). Like MYB2, MYB62 is a member of the MYB-R2R3 family and localizes in the nucleus (Table 1; Devaiah et al., 2009). Under Pi-sufficient and Pi-deficient conditions, the transcript levels of gibberellic acid (GA) biosynthetic genes and PSI genes decrease in MYB62-overexpressing plants, which have a GA-deficient phenotype. MYB2 is a transcriptional activator, whereas MYB62 suppresses target gene transcription during stress (Devaiah et al., 2009).

WRKY transcription factors

WRKY transcription factors are involved in auto-regulation and cross-regulation by modulating plant transcriptional processes in multiple stress signaling pathways (Banerjee and Roychoudhury, 2015; Phukan et al., 2016). WRKY transcription factors with a C2H2 zinc finger domain control target gene transcription by binding to W box (TTGACT/C) elements (Chiou and Lin, 2011; Rushton et al., 2010). The WRKY6 transcription factor is a typical WRKY family member with roles in the responses to different stimuli, where it enhances the PR1 promoter activity in senescence and pathogen-defense signaling (Chen et al., 2009; Huang et al., 2016; Robatzek and Somssich, 2002). WRKY6 expression is also highly induced by bacterial pathogens and it increases the senescence-induced receptor-like kinase promoter’s activity in response to the bacterial elicitor flagellin (Robatzek and Somssich, 2002). WRKY6 directly binds to the W box within the RAV1 promoter and decreases its gene transcript level during ABA stress response (Huang et al., 2016). Thus, WRKY6 modulates the cross-talk among different stress responses by regulating the transcription of various target genes (Table 1).

WRKY6 negatively regulates PHO1 expression (Chen et al., 2009) and a WRKY6 homolog, WRKY42, positively regulates Pht1 and PHO1 transcription in the Pi starvation response (Table 1; Su et al., 2015). WRKY6 and WRKY42 are both degraded via 26S proteasome-mediated proteolysis in the Pi starvation response (Chen et al., 2009; Su et al., 2015). WRKY45 is specifically expressed in roots and binds to two W box elements in the promoter of Pht1 to regulate its transcription (Table 1; Wang et al., 2014). A root hair-specific WRKY75 affects transcriptional cross-talk among Pi starvation, phytohormones, and biotic stress signaling pathways (Table 1). WRKY75 mutation suppresses the transcription of PSI genes, including phosphatases, Mt4/TPS1-like genes, and Pi transporters (Devaiah et al., 2007a). WRKY75 overexpression increases the transcript levels of jasmonic acid (JA) marker genes, such as PDF1.2, VSP1, and LOX2, but it decreases the expression of PR1, a salicylic acid (SA) marker gene (Chen et al., 2013; Schmiesing et al., 2016). Interestingly, WRKY45 and WRKY75 are mutual negative regulators in auto-regulation, where WRKY75 represses WRKY45 gene transcription by binding two W box elements within the WRKY45 promoter (Wang et al., 2014).

Other transcription factors

There are numerous other transcription factors that are important components of the transcriptional regulatory system of stress-responsive genes (Nakashima et al., 2009). C2H2-type zinc finger protein transcription factors function as essential components in Pi starvation and other abiotic stresses (Sakamoto et al., 2000). ZAT6 binds to three different sequences of POS9 (P-INO-specific regions) motifs in target gene promoters during developmental processes and the Pi starvation response (Table 1; Devaiah et al., 2007b; Meister et al., 2004). ZAT6 is strongly induced and closely related to abiotic stress responses, such as salt, cold, osmotic, and drought stresses, by binding to DRE (dehydration-responsive element) in target gene promoter regions (Table 1; Liu et al., 2013; Vogel et al., 2005). ZAT6 is highly expressed under cold stress and it regulates CBF2 transcription by binding to DRE within its promoter (Vogel et al., 2005).

IN SILICO ANALYSIS OF PUTATIVE CIS-ACTING REGULATORY ELEMENTS IN PI-RESPONSIVE GENE PROMOTERS

Phosphate transporters

Plants have diverse biological mechanisms for enhancing the availability of external Pi in the soil via Pi transporters (Chiou and Lin, 2011; Raghothama, 2000). Pi transporters are encoded by members of PHT gene families, including nine Pht1 members, one Pht2 member, three Pht3 members, six Pht4 members, and three Pht5 members in Arabidopsis (Guo et al., 2008; Knappe et al., 2003; Liu et al., 2016; Rausch and Bucher, 2002). Pht1;1 transcription is positively regulated by PHR1 (Rubio et al., 2001), WRKY75 (Devaiah et al., 2007a), WRKY45 (Wang et al., 2014), and WRKY42 (Su et al., 2015) but negatively regulated by MYB62 (Devaiah et al., 2009) under Pi-deficient conditions. Several types of cis-acting regulatory elements exist in the Pht1;1 promoter, such as P1BS, W box, and MBS. To understand the transcriptional regulation of Pi transporters, we conducted in silico analysis based on the DNA sequences of Pi transporter promoter regions and showed that the expression of Pi transporters could be regulated by hormones and various other stresses as well as by Pi starvation (Tables 2 and 3).

Table 2

Analysis of hormone signaling-related putative cis-acting regulatory elements in Pi starvation-responsive gene promoters

Stress	Motif Name	Sequence	Gene Name (Number of sites in the promoter)
			AtPTs	microRNAs	PSI
Auxin	AuxRE	TGTCTCAATAAG	AtPht1;8(1)	miR2111a(1)	None
	AuxRR-core	GGTCCAT	AtPht1;9(1), AtPht4;1(1)	miR156g(2)	SPX1(1), LPR1(1)
	TGA-element	AACGAC	AtPht1;4(2), AtPht1;7(2), AtPht3;1(1), AtPht3;2(1), AtPht4;1(1), AtPht4;5(1), AtPht4;6(1), AtPht5;2(1), AtPht5;3(1)	miR156c(2), miR156g(1), miR156h(1), miR2111a(1)	SPX4(2), PHR1(2), SCR(1), PAP2(1)
		TGACGTAA	None	miR156b(1)	None
	TGA-box	TGACGTGGC	None	miR2111b(1)	None
Ethylene	ERE	ATTTCAAA	AtPht1;3(1), AtPht1;4(2), AtPht1;6(1), AtPht3;1(1), AtPht3;3(1), AtPht4;2(1)	miR156a(2), miR156b(1), miR156c(1), miR156e(2), miR2111b(1)	At4/IPS2(1), PAP2(1)
GA	P-box	CCTTTTG	AtPht1;4(2), AtPht1;5(1), AtPht1;8(2), AtPht4;6(1)	miR156b(1), miR156c(1), miR2111a(2)	SPX2(1), PHR1(1), RNS1(1), At4/IPS2(2), PDR2(2), LPR2(1), SCR(1), BAH1(1)
		GCCTTTTGAGT	None	miR399d(1), miR399e(1)	IPS1(1)
	GARE-motif	TCTGTTG	AtPht1;2(1), AtPht1;4(1), AtPht1;5(1), AtPht1;7(1), AtPht1;8(1), AtPht1;9(1), AtPht3;2(2), AtPht4;5(1), AtPht4;6(1), AtPht5;2(2)	miR156b(1), miR156e(1), miR399b(1), miR399e(2), miR778a(1), miR827a(1)	SPX3(1), PHO2(1)
		AAACAGA	AtPht1;1(1), AtPht1;3(1), AtPht1;4(1), AtPht1;7(3), AtPht1;8(2), AtPht1;9(2), AtPht3;1(1), AtPht4;1(1), AtPht4;2(1), AtPht4;6(1), AtPht5;1(1)	miR156c(2), miR156d(2), miR399b(1), miR399c(1), miR778a(2), miR827a(3)	PHR1(1), PHF1(2), PHO2(5), LPR2(2), SCR(3), BAH1(4)
	TATC-box	TATCCCA	AtPht4;1(2), AtPht4;5(1), AtPht5;3(1)	miR156e(1), miR156h(1), miR778a(1)	SPX3(1), BAH1(1)
JA	CGTCA-motif	CGTCA	AtPht1;1(1), AtPht1;4(2), AtPht1;5(2), AtPht1;6(2), AtPht1;7(3), AtPht1;9(2), AtPht3;1(1), AtPht3;2(4), AtPht3;3(3), AtPht4;1(2), AtPht4;2(1), AtPht4;3(1), AtPht4;4(2), AtPht4;5(2), AtPht4;6(1), AtPht5;3(1)	miR156b(3), miR156c(2), miR156d(1), miR156g(2), miR156h(4), miR399c(1), miR399d(1), miR399f(1), miR778a(3), miR827a(1), miR2111b(2)	SPX1(2), SPX3(1), SPX4(2), PHR1(2), PHF1(1), PHO1(2), PHO2(3),SIZ1(1), PDR2(2), LPR1(2), SCR(2), PAP2(2)
	TGACG-motif	TGACG	AtPht1;1(1), AtPht1;4(2), AtPht1;5(2), AtPht1;6(2), AtPht1;7(3), AtPht1;9(2), AtPht3;1(1), AtPht3;2(4), AtPht3;3(3), AtPht4;1(2), AtPht4;2(1), AtPht4;3(1), AtPht4;4(2), AtPht4;5(2), AtPht4;6(1), AtPht5;3(1)	miR156b(3), miR156c(2), miR156d(1), miR156g(2), miR156h(4), miR399c(1), miR399d(1), miR399f(1), miR778a(3), miR827a(1), miR2111b(2)	SPX1(2), SPX3(1), SPX4(2), PHR1(2), PHF1(1), PHO1(2), PHO2(3),SIZ1(1), PDR2(2), LPR1(2), SCR(2), PAP2(2)
SA	SARE	TTCGACCATCTT	AtPht3;3(1), AtPht5;3(1)	None	None
	TCA-element	CCATCTTTTT	AtPht1;4(1), AtPht2;1(1), AtPht3;1(1), AtPht4;6(2), AtPht5;1(1), AtPht5;3(2)	miR156c(1), miR156e(1), miR156f(1), miR399b(2), miR399c(1), miR2111b(1)	SPX1(1), SPX3(1), SPX4(1), PHO1(1), RNS1(3), IPS1(1), SIZ1(1), PDR2(1), SCR(3)
		GAGAAGAATA	AtPht1;1(1), AtPht1;2(1), AtPht1;3(1), AtPht1;4(1), AtPht1;6(1), AtPht1;7(2), AtPht1;8(1), AtPht1;9(1), AtPht2;1(1), AtPht4;1(2), AtPht4;4(2), AtPht5;1(1), AtPht5;2(1), AtPht5;3(1)	miR156a(1), miR156d(1), miR156e(1), miR399c(1), miR827a(3), miR2111a(1), miR2111b(1)	SPX3(1), SCR(1), PAP2(1)
		CAGAAAAGGA	AtPht2;1(1), AtPht3;1(1), AtPht3;3(1), AtPht4;3(1)	miR156d(1)	LPR1(1), SCR(1)
		TCAGAAGAGG	AtPht1;4(1), AtPht2;1(1)	miR156e(1), miR2111b(1)	None

In silico analysis was conducted using 1.5 kb upstream promoter regions from first exon start site of each gene by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Table 3

Analysis of various stresses signaling-related putative cis-acting regulatory elements in Pi starvation-responsive gene promoters

Stress	Motif Name Sequence		Gene Name (Number of sites in the promoter)
			AtPTs	microRNAs	PSI
ABA	ABRE	ACGTGGC	AtPht4;1(1), AtPht4;4(1)	miR2111b(1)	LPR1(1)
		AGTACGTGGC	None	miR399e(1)	None
		CACGTG	AtPht4;1(1), AtPht4;2(1), AtPht4;3(1), AtPht4;4(1), AtPht4;5(1)	miR156b(2), miR156c(1), miR156e(1), miR156h(1), miR399e(1), miR2111a(1)	SIZ1(1)
		CGCACGTGTC	None	miR2111a(1)	None
		GCAACGTGTC	AtPht5;1(1), AtPht5;3(1)	miR156d(1)	None
		GCCACGTACA	AtPht3;3(1)	None	None
		GCCGCGTGGC	AtPht4;1(1), AtPht4;2(1)	None	BAH1(1)
		TACGTG	AtPht1;1(1), AtPht1;2(1), AtPht1;3(1), AtPht3;2(1), AtPht3;3(1), AtPht4;1(1), AtPht4;2(1), AtPht4;4(1), AtPht4;6(1), AtPht5;1(1)	miR156a(1), miR156d(1), miR156h(1), miR399c(1)	SPX1(1), SPX3(1), PHR1(1), PHF1(1), PHO1(1), RNS1(3), IPS1(2), SIZ1(1), LPR10(1)
		TACGGTC	None	miR778a(1), miR827a(1)	SIZ1(1)
	CE3	GACGCGTGTC	None	miR156h(1)	None
Drought	C-repeat/DRE	TGGCCGAC	AtPht1;9(1)	None	None
	MBS	CAACTG	AtPht1;8(1), AtPht3;2(1), AtPht4;2(1), AtPht5;1(1), AtPht5;3(1)	miR156e(1), miR399a(1), miR399c(1)	SPX2(1), PHO2(3), At4/IPS2(1), SCR(1), PAP2(2)
		CGGTCA	AtPht1;4(1), AtPht4;5(1)	miR156h(1), miR399b(1), miR778a(1), miR827a(2)	SPX2(1), SPX3(1), PHO2(1)
		TAACTG	AtPht1;1(1), AtPht1;3(1), AtPht1;4(2), AtPht1;5(1), AtPht1;8(1), AtPht2;1(1), AtPht4;2(1), AtPht4;3(3)	miR399c(1), miR399d(2), miR399f(2)	SPX1(1), SPX2(4), PHO1(2), PHO2(2), LPR1(1), LPR2(1)
Cold	LTR	CCGAAA	AtPht1;5(1), AtPht1;6(1), AtPht1;8(2), AtPht2;1(3), AtPht3;1(2), AtPht3;3(1), AtPht4;2(1), AtPht4;5(1), AtPht4;6(1), AtPht5;2(1)	miR156c(1), miR156d(1), miR156e(1), miR156f(1), miR156g(2), miR399d(1), miR399f(1), miR827a(1)	SPX1(2), SPX4(2), PHR1(1), PHO1(1), PHO2(1), SIZ1(2), PDR2(2), LPR2(1)
Defense and stress	TC-rich repeats	ATTCTCTAAC	AtPht1;9(2), AtPht5;3(1)	miR156c(1), miR156e(1), miR156f(1), miR827a(1), miR2111a(1)	LPR2(1)
		ATTTTCTTCA	AtPht1;7(2), AtPht2;1(4), AtPht3;1(1), AtPht3;2(1), AtPht4;1(1), AtPht4;2(1), AtPht4;5(1), AtPht5;2(3), AtPht5;3(3)	miR156b(1), miR156f(1), miR156h(1), miR399b(1), miR399c(1), miR399d(1), miR399e(2)	SPX1(1), SPX2(2), SPX4(1), PHR1(2), At4/IPS2(1), SIZ1(1), PDR2(1), LPR2(1)
		ATTTTCTCCA	AtPht1;1(1), AtPht1;5(1), AtPht1;6(1), AtPht3;2(2), AtPht4;1(1), AtPht4;3(3), AtPht5;2(1)	miR778a(1)	PHR1(1), PHF1(1), LPR2(1)
		GTTTTCTTAC	AtPht1;2(1), AtPht1;3(1), AtPht1;4(1), AtPht1;6(1), AtPht1;7(1), AtPht4;3(1), AtPht5;2(1)	miR156c(1), miR156e(1), miR156h(1), miR399c(1), miR778a(2), miR2111b(1)	IPS1(1), At4/IPS2(2), SCR(2)
Fungal	Box-W1	TTGACC	AtPht1;1(2), AtPht1;3(1), AtPht1;6(1), AtPht1;9(1), AtPht3;1(3), AtPht3;3(1), AtPht4;3(1), AtPht4;4(2), AtPht4;6(1), AtPht5;3(1)	miR156c(2), miR156h(1), miR399a(1), miR399e(1), miR827a(1)	SPX1(1), SPX2(1), SPX3(1), SPX4(1), SCR(1), BAH1(1)
Heat	HSE	AGAAAATTCG	AtPht1;7(2), AtPht3;2(1), AtPht5;1(1)	miR156b(1), miR156g(2), miR399a(1)	SPX2(3), SPX3(1), PDR2(1), LPR1(1), SCR(1)
		AAAAAATTTC	AtPht1;1(3), AtPht1;3(1), AtPht1;4(1), AtPht1;6(2), AtPht1;7(3), AtPht1;8(1), AtPht1;9(1), AtPht2;1(3), AtPht3;2(1), AtPht4;1(1), AtPht4;2(1), AtPht4;4(1)	miR156a(3), miR156b(1), miR156f(1), miR156g(1), miR399b(2), miR399c(1), miR778a(2), miR2111a(2)	SPX2(1), PHR1(1), PHF1(1), PHO1(2), RNS1(4), At4/IPS2(1), PDR2(2), LPR1(1), SCR(1)
		CNNGAANNTTCNNG	AtPht1;9(1)	None	None
Wound	WUN-motif	TCATTACGAA	AtPht1;4(1), AtPht3;1(1), AtPht4;1(1), AtPht4;6(1)	miR399c(2)	SPX3(1), PHO1(1), BAH1(1), PAP2(1)

In silico analysis was conducted using 1.5 kb upstream promoter regions from first exon start site of each gene by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

In silico analysis suggest that Pht1;4, Pht1;7, Pht1;8, Pht1;9, Pht3;1, Pht3;2, Pht4;1, Pht4;5, Pht5;2 and Pht5;3 gene transcription is possibly regulated by auxin because their promoters contain auxin-related putative cis-acting regulatory elements such as AuxRE, AuxRR-core, TGA-element, and TGA-box (Table 2). The Pht1;3, Pht1;4, Pht1;6, Pht3;1, Pht3;3 and Pht4;2 gene promoters contain ethylene-responsive cis-acting elements, and a GA-responsive element is found in most Pi transporter genes except Pht1;6, Pht2;1, Pht3;3, Pht4;3, and Pht4;4 (Table 2). A previous report shows the induction of Pht1;4 expression by ethylene supporting the reliability of our in silico analysis for understanding the regulation of Pi starvation-responsive gene networks by other stresses (Lei et al., 2011b). Most Pi transporters contain putative cis-acting regulatory elements in their promoters, such as CGTCA-motif, TGACG-motif, SARE, and TCA-element, which are related to SA- and JA-mediated plant defense signaling (Table 2). The ABA or drought stress-responsive elements ABRE, DRE, and MBS also exist in most Pi transporters, except Pht1;6, Pht1;7, Pht3;1, and Pht5;2, and the cold-responsive element LTR is found in the Pht1;5, Pht1;6, Pht1;8, Pht2;1, Pht3;1, Pht3;3, Pht4;2, Pht4;5, Pht4;6, and Pht5;2 gene promoters (Table 3). Many Pi transporters have TC-rich repeats related to defense and stress responses, except the Pht1;8, Pht3;3, Pht4;4, Pht4;6, and Pht5;1 genes, and an HSE element for heat stress response, except the Pht1;2, Pht1;5, Pht3;1, Pht4;3, Pht4;5, Pht4;6, Pht5;2, and Pht5;3 genes (Table 3). Fungal stress–related Box-W1 elements are found in the Pht1;1, Pht1;3, Pht1;6, Pht1;9, Pht3;1, Pht3;3, Pht4;3, Pht4;4, Pht4;6, and Pht5;3 genes, and wounding stress-related WUN-motifs are predicted in the Pht1;4, Pht3;1, Pht4;1, and Pht4;6 gene promoters (Table 3).

microRNAs

Many microRNAs (miRNAs) such as miR156, miR399, miR778, miR827, and miR2111, are major regulators in Pi starvation signaling (Chiou et al., 2006; Hsieh et al., 2009; Pant et al., 2009). We showed that miR399f expression is regulated by the MYB2 transcription factor, which has roles in salt, ABA, and drought stress signaling (Table 1; Abe et al., 2003; Yoo et al., 2005) by directly binding to the MBS element in the miR399f precursor promoter (Baek et al., 2013). Moreover, salt and ABA stress enhance the activity of the miR399f promoter (Baek et al., 2016). The miR399f precursor promoter contains several cis-acting regulatory elements, such as CGTCA-motif (involved with JA) and LTR (linked with cold stress) (Baek et al., 2013). miR156 is a key player in the Pi starvation response and flowering, and it also plays an important role in salt, drought, and heat stress signaling (Cui et al., 2014; Stief et al., 2014). The transcription of miR156c is rapidly and greatly induced in response to salt and drought stresses via MYC, ERF, and W box motifs in the miR156c precursor promoter (Cui et al., 2014). Our in silico analysis showed that the miR156c precursor promoter contains various cis-acting elements, such as TGA-element, P-box, GARE-motif, CGTCA-motif, TCA-element, ABRE, LTR, TC-element, and Box-W1, thereby suggesting cross-talk between Pi starvation and various types of stress signaling during the regulation of miRNAs (Tables 2 and 3).

Phosphate starvation-inducible genes

The expression of many Pi starvation-responsive genes is cross-regulated by Pi starvation and other stress signaling pathways. Plant phytohormones, such as cytokinin, ethylene, ABA, and auxin are associated with the transcription of genes involved in the Pi starvation response. PHO1 plays a crucial role in Pi starvation signaling and it is significantly down-regulated by auxin, cytokinin, and ABA (Ribot et al., 2008). RNS1 is a secreted ribonuclease and another Pi starvation–related gene that is significantly upregulated by ABA (Hillwig et al., 2008). The RNS1 promoter contains several putative cis-acting elements, including ABRE, MYB/MYC, WUN-motif, W box, HSE, P-box, and TCA elements, which mediate various stress signaling pathways (Tables 2 and 3; Hillwig et al., 2008). SIZ1 is a small ubiquitin-like modifier E3 ligase paying important roles in enhancing the tolerance of environmental stresses such as salt, cold, drought, ABA, auxin, SA, and Pi starvation (Catala et al., 2007; Miura et al., 2005; 2007; 2009; 2010; 2011a; 2011b). Multiple functions of SIZ1 are known in various stress signaling pathways, but the transcriptional regulation of its expression remains unknown. Our in silico analysis indicates that the SIZ1 promoter contains various putative cis-acting regulatory elements, such as ABRE, LTR, TC-rich repeats, WUN-motif, CGTCA-motif, and TCA-element, which function in diverse stress signal transduction cascades (Tables 2 and 3). Our results provide biological insights into the mechanisms that regulate SIZ1 expression as well as its biological functions in plant stress responses. In summary, findings of our in silico analysis of the regulatory regions of Pi starvation–related genes, such as Pi transporters, miRNAs, and PSI genes, suggest that their expression may be related to various environmental stresses to maintain Pi homeostasis in plants.

CONCLUSION

Phosphorus in the form of Pi is an essential nutrient for plant growth, development, and productivity, but Pi is one of the least available essential nutrients because of its insolubility and low available concentrations (Poirier and Bucher, 2002; Raghothama, 1999). To cope with Pi starvation, plants reprogram various cellular processes, including the reduction of internal Pi usage and activation of external Pi acquisition and recycling. Studies on Pi starvation signaling in plants have identified signaling components, such as transcription factors, non-coding RNAs, and protein modifiers, but also cross-talk with other plant signaling pathways including phytohormones, sugars, and other nutrients (e.g., iron) (Rouached et al., 2010; Yuan and Liu, 2008). Biotic and abiotic stresses significantly affect plant growth, but the links between Pi starvation and other environmental stress signaling pathways remain unclear. Understanding the cross-regulation of gene expression by identifying the transcription factors involved in both Pi starvation and diverse environmental stress signaling pathways, as well as in silico analysis of cis-acting elements in the regulatory regions of Pi starvation signaling components, will provide molecular mechanisms of the connections between Pi starvation and other environmental stress signaling pathways.