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BMC Genomics

Open Access

Transcriptional analysis of phloem-associated cells of potato

  • Tian Lin1,
  • Coralie C. Lashbrook1,
  • Sung Ki Cho1,
  • Nathaniel M. Butler1, 2,
  • Pooja Sharma1,
  • Usha Muppirala3,
  • Andrew J. Severin3 and
  • David J. Hannapel1Email author
BMC Genomics201516:665

https://doi.org/10.1186/s12864-015-1844-2

Received: 16 February 2015

Accepted: 14 August 2015

Published: 3 September 2015

Abstract

Background

Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species.

Results

Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome.

Conclusions

As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

Keywords

Laser capture microdissectionMobile RNAPhotoperiodPolypyrimidine tract bindingRNA-binding proteinsSieve elementsSignalVascular biology

Background

Plants are sessile organisms and unlike animals have no neural network or circulatory system. Phloem and xylem are the main tissues that facilitate nutrient and signal transport in the whole-plant body. With the evolution in size and complexity, the need for an efficient long-distance transport system has steadily increased over time for land plants [1]. The result of these changes has led to the development of more specialized and complicated cell types in both the phloem and xylem. Xylem is composed of parenchyma cells, fibers and long tracheary elements that transport water and soluble mineral ions from the root to other organs. Tracheary elements and fibers are enucleate, non-living cells that maintain only a cell wall. In comparison, phloem is composed of living cell types, including sieve elements, parenchyma cells, and supportive cells, such as fibers and sclereids. Parenchyma cells include both specialized companion cells and unspecialized phloem parenchyma cells. Sieve elements lose most of their organelles and are enucleate. All their metabolic functions are carried out by the companion cells but profiles of phloem proteins suggest that translation may occur within the sieve element system [2]. RNAs are transcribed and translated in companion cells and small RNAs, mRNAs and proteins are then actively transported into sieve elements through the plasmodesmata [3].

Phloem is the conduit for transport of photosynthates, mainly sucrose, from leaf to sink tissues. Signal molecules also take advantage of this information highway to communicate between different organs. These molecules can be hormones [4], small RNAs [5], full-length mRNAs [612] and proteins like FT [13, 14]. From numerous studies of phloem-mobile signals, it is clear that these molecules can be delivered in either an acropetal or basipetal direction. Two examples illustrate how such phloem-mobile signals regulate development [15]. Under photoperiodic conditions inductive for flowering, FLOWERING LOCUS T (FT) is expressed in the leaf and transported in protein form through the sieve element system to the shoot apex where, in conjunction with FLOWERING LOCUS D (FD), it activates the floral pathway [16]. Several studies have identified FT in the shoot apex or phloem exudate of plants induced for flowering [13, 17, 18]. In this system, FD provides spatial control of flowering and FT provides temporal control. As another example, using heterografting experiments, full-length StBEL5 mRNA of potato was verified to move in a downward direction from leaf to stolon and root [9, 19]. This long-distance phloem transport of StBEL5 is enhanced under short days and controlled by untranslated regions of the transcript. Movement of StBEL5 mRNA was correlated with increased tuber yields and root growth [9, 19]. Both of these long-distance signaling systems utilize photoperiodic cues to activate movement of the developmental signal from source to sink organs.

The mechanism of non-cell autonomous movement and its regulation are still unclear, but RNA-binding proteins (RBP) identified from phloem sap of pumpkin bind to mobile mRNAs to regulate their movement [2, 20]. A polypyrimidine tract-binding (PTB) protein of pumpkin (RBP50) was identified as the core protein of a RNA/protein complex that transports RNA. Further evidence suggests that similar RBPs in potato function to facilitate both stability and long-distance transport of select mobile RNAs [21]. Transcription of these RBPs was observed in companion cells of the phloem [20]. To elucidate the potential for long-distance signaling through the sieve element system, several profiles of phloem proteins and RNAs have been undertaken. Analysis of the proteome of phloem sap of pumpkin revealed over 1200 proteins present in the sieve element system [2]. Through both phloem cell microdissection and analysis of phloem sap, we now know that the transcriptome of phloem includes thousands of full-length mRNAs with a diverse range of potential functions [22, 23]. Phloem sap, in particular, has been used as an efficient source to study uncontaminated phloem proteins and RNAs [2, 20, 24]. Results from the most widely used model system for phloem sap analysis, the cucurbits, have been compromised, however, due to the existence of two separate phloem sources each with unique protein and RNA sets [25]. In most plant species pure phloem sap cannot be obtained. As a reasonable alternative, laser capture microdissection (LCM) makes it possible to isolate RNA from specific cell types and provides practical access to expression profiles of phloem tissue [2629]. In previous studies, transcripts of seven of the StBEL genes of potato, including the mobile mRNA, StBEL5, were identified in RNA extracted from phloem cells using LCM/RT-PCR [30]. Combining LCM and RNA-seq has proven to be an invaluable tool for profiling high-resolution transcription in specific cells [28, 3134]. Here we make use of LCM and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. The combination of these techniques has provided a valuable approach to the study of phloem tissue and a comprehensive snapshot into the mechanisms associated with long-distance signaling.

Results

Analysis of a LCM phloem transcriptome

To gain insight into the function of the numerous genes actively involved in transport and signaling throughout the phloem system, transcriptomes of phloem-associated cells (PAC) were profiled from both the petiole and the lower stem of short day-grown potato plants. The petiole was selected because of its proximity to the leaf, an important source of a wide range of light-activated and photosynthate-related signals. The lower stem was selected because of its proximity to the strong tuber sink. RNA was isolated from phloem tissue samples dissected from paraffin-embedded petiole (Short day [SD] Petiole-phloem) and stem sections (SD Stem-phloem) using the LCM method [30]. Because of our interest in the short day (SD) activated process of tuber formation in potato, both samples come from SD-grown plants. The sample collected by LCM contains not only sieve elements, but also the companion cells, phloem parenchyma cells and other cells associated with the phloem. Making use of a phloem-specific marker, StPTB1 [35], phloem cells that were harvested can be observed in scattered bundles in the petiole (Fig. 1a-c) and in outer regions of discrete vascular bundles in the stem (Fig. 1d). Based on this morphology, the transcriptome profiled from the LCM-derived samples represents the transcriptome of PAC. RNA yields from LCM-derived samples are commonly very low. To obtain a working concentration, extracted RNA was amplified and three replicates per tissue type were analyzed.
Fig. 1

Transverse section of potato petiole and stem to show phloem tissue collected in LCM with phloem specific marker StPTB1. Localization of GUS activity within petioles (a-c) and stems (d) of plants that contained the StPTB1 promoter driving GUS expression [35]. Petiole and stem internodes of tuberizing 4-week old soil grown plants were embedded in paraffin for histochemical detection of GUS activity within tissues of the petiole and stem. Panel (a) is a transverse section of StPTB1prom activity with a higher magnification image of a vascular bundle (boxed area) in (b-c) showing abaxial-side phloem cells. Panel (d) is a transverse section of StPTB1prom activity in the vascular bundle of a stem showing external (EP) phloem tissues

The number of reads contained in each library was greater than 2.9 × 107 and only 25 to 46.9 % reads of the reads were mapped to the genome uniquely, where both pairs of reads mapped in the expected direction and with the expected distance between them (concordantly). Most of the other reads were mapped to multiple locations in the genome. Of the approximately 39 k genes contained in the potato genome, 15 to 23 k genes were detected in the three replicates of phloem-associated cells (either petiole-PAC or stem-PAC) (Additional file 1: Table S1). Most of the genes that were expressed in only one or two of the replicates are present at very low abundance (<10 reads, for example). The six libraries were normalized with the upper quantile and analyzed with a Generalized Linear Model using QuasiSeq. P-values were calculated with QLSpline based on negative binomial distribution. Q-values were given by adjusting the p-value for familywise error rate (Additional file 2: Figure S1). After removing the effect of the sequencing method, a mean value was calculated for petiole-PAC and stem-PAC to indicate their measured level. All of the genes identified in the transcriptomes of this report with mean read values are listed in Additional file 3: Table S2. Excluding the low read hits (<10 reads), 15,167 genes can be identified in either petiole-PAC or stem-PAC transcriptome (Group 2 under the whole genome column, Table 1). The number of expressed genes are comparable to the 14,242 and 13,775 genes identified in the vascular bundles of cucumber and watermelon, respectively [36].
Table 1

Distribution of gene expression in different gene ontology categories for both differentially expressed (Group 1) and unique genes of petiole- and stem-PACs

GO terms were searched from the AmiGO 2 GO browser. GO analysis of the potato genome was performed with Blast2GO. Light-related and hormone-related were excluded from the signaling-related category

Comparison of Petiole-PAC and stem-PAC transcriptomes

Out of the 26,898 genes that exhibited any expression in either petiole-PAC or stem-PAC, 2087 were identified as differentially expressed (DE) genes between petiole-phloem and stem-phloem, with q-values less than 0.05 (Additional file 4: Table S3). Most of these genes are expressed at low abundance levels (<10 mean value in both petiole-PAC and stem-PAC). Only 573 DE genes have a mean value >10 in either petiole-PAC or stem-PAC (Additional file 4: Table S3). At the 500 read cutoff level, the number of DE genes between petiole-PAC and stem-PAC is only 162 (Table 1, Group 1). This suggests that the petiole PAC and stem PAC have very similar transcriptomes.

To visualize functional relationships in these expression profiles, an ontological analysis was performed. Gene ontology (GO) categories of all the genes in potato were obtained from the GO database using Blast2GO, with parameters of 20 hits and an e-value of 10e−6. 22,058 genes out of the 39,028 genes (56.5 %) in the potato genome were matched with at least one GO term. The 573 DE genes were mapped to 3791 GO terms including 736 molecular functions, 2579 biological processes, and 476 cellular components (Fig. 2). As we would expect with active transport and loading through the phloem cells, binding function is one of the most prominent functions in the genes expressed in PAC. Out of the 736 molecular functions, 81, 62, and 126 were classified as “nucleotide binding”, “protein binding”, and “binding”, respectively (Fig. 2). “DNA binding” and “RNA binding” have lower numbers, 33 and 16 respectively, but these functions have very important roles in transcription, mRNA stabilization, localization and transport (Fig. 2, arrows). The signaling-related biological processes and molecular functions were more closely examined for all unique and DE genes related to signal transport, a prominent function of phloem (Table 1). Specifically, the transcripts encoding proteins functional in signaling and regulation, such as light-induced signaling, photoperiodism, floral induction, hormone-related signaling and transcription factors, were explored due to their importance in long-distance transport. Among these DE “signal” genes abundantly expressed, 10 are classified as signaling-related, 20 as light-related, 5 as hormone-related, and 11 as flowering-related (Group 1, Table 1). Eight genes are classified as transcription factors and six encode for RNA-binding proteins. DE genes from signaling, light, hormone, flowering, and RNA-binding GO categories are listed in Table 2.
Fig. 2

Distribution of molecular functions in the differentially expressed genes between petiole-PAC and stem-PAC. Differentially expressed (DE) genes were identified with generalized linear model by using QuasiSeq [93]. Genes with less than ten reads in both petiole-PAC and stem-PAC were removed because of their low abundance. GO terms of the whole genome were analyzed with Blast2GO. The GO categories identified for each gene were made comparable by converting each GO term to the same level in the GO structure to permit. This was done with goslimviewer in AgBase [96] (http://agbase.msstate.edu/cgi-bin/tools/goslimviewer_select.pl)

Table 2

Differentially expressed genes between petiole-PAC vs. stem-PAC with select gene ontology categories

Related function

Signaling-related genes

Petiole phloem

Stem phloem

Long day mean

Short day mean

Organ effect. Q-value

Photoperiod Q-value

Annotation

Signaling

PGSC0003DMG400000584

1211

366

486

1487

1.99E-02

6.80E-10

Pseudo-response_regulator_5

Signaling

PGSC0003DMG400000792

1337

335

775

710

3.12E-02

7.75E-02

Ran_GTPase_binding_protein

Signaling

PGSC0003DMG400001272

449

1474

15,250

16,791

4.78E-02

1.69E-02

ADP-ribosylation_factor_1

Signaling

PGSC0003DMG400002613

1949

941

3162

3697

2.42E-02

7.82E-02

PDR8_PEN3_(PLEIOTROPIC_DRUG_RESISTANCES)

Signaling

PGSC0003DMG400005792

115

504

114

112

1.57E-02

3.37E-01

Nucleoside_diphosphate_kinase

Signaling

PGSC0003DMG400016822

761

55

360

340

1.30E-02

1.45E-01

ADP-ribosylation_factor

Signaling

PGSC0003DMG400017350

62

604

454

452

3.29E-02

3.65E-01

ATP binding_protein

Signaling

PGSC0003DMG400021253

1936

691

2978

2746

3.21E-02

6.53E-02

Mitogen-activated_protein_kinase

Signaling

PGSC0003DMG400023211

188

538

2670

4268

2.75E-02

2.65E-07

Phospholipase_C

Signaling

PGSC0003DMG400028694

1132

330

1330

975

4.05E-02

1.67E-05

Ethylene receptor homolog

Light

PGSC0003DMG400000584

1211

366

486

1487

1.99E-02

6.80E-10

Pseudo-response_regulator_5

Light

PGSC0003DMG400000792

1337

335

775

710

3.12E-02

7.75E-02

Ran_GTPase_binding_protein

Light

PGSC0003DMG400001299

41

883

1418

1372

3.29E-02

2.11E-01

Ankyrin_repeat_domain_protein

Light

PGSC0003DMG400001342

894

149

1299

1288

3.44E-04

3.60E-01

Conserved_gene_of_unknown_function

Light

PGSC0003DMG400005792

115

504

114

112

1.57E-02

3.37E-01

Nucleoside_diphosphate_kinase

Light

PGSC0003DMG400007966

237

630

1991

2222

1.15E-02

1.11E-02

Annexin

Light

PGSC0003DMG400008589

705

354

2624

2510

2.15E-02

9.22E-02

RNA_helicase

Light

PGSC0003DMG400010794

322

1756

3137

2419

3.40E-02

6.32E-05

Cellulose_synthase

Light

PGSC0003DMG400018104

2304

870

2852

1442

4.36E-02

6.46E-10

Cell_division_cycle_protein_48

Light

PGSC0003DMG400018449

5071

8343

15,036

18,618

3.26E-02

2.70E-05

Actin

Light

PGSC0003DMG400018795

1366

33

79

100

1.22E-02

3.01E-02

Multidrug_resistance_protein_1,_2

Light

PGSC0003DMG400020086

1604

601

3090

3130

3.00E-02

3.16E-01

26S_proteasome_subunit_4

Light

PGSC0003DMG400022381

1357

650

2758

3376

2.39E-03

1.07E-04

Conserved_gene_of_unknown_function

Light

PGSC0003DMG400024249

782

1654

459

835

7.52E-03

1.02E-08

Gibberellin_20-oxidase-l

Light

PGSC0003DMG400026500

322

792

17,945

19,242

4.43 E-02

5.04E-02

Type_1_(26_kD)_CP29_polypeptide

Light

PGSC0003DMG400029829

2102

3550

9228

8869

1.07E-02

9.70E-02

Eukaryotic_initiation_factor_3E_subunit

Light

PGSC0003DMG400030867

1073

2924

6003

5469

1.67E-02

5.99E-02

Acyl-CoA-binding_protein

Light

PGSC0003DMG400031124

611

1050

2412

2959

4.85E-02

2.95E-02

Myosin_Xl-F

Light

PGSC0003DMG400031812

60

515

1556

1554

2.89E-02

3.70E-01

DNA_photolyase

Light

PGSC0003DMG400035320

18

668

222

221

1.92E-03

3.70E-01

F-box_leucine_rich_repeat_protein

Hormone

PGSC0003DMG400001342

894

149

1299

1288

3.44E-04

3.60E-01

Conserved_gene_of_unknown_function

Hormone

PGSC0003DMG400009773

540

1364

2414

2546

1.50E-02

2.34E-01

Auxin_response_factor_19

Hormone

PGSC0003DMG400010794

322

1756

3137

2419

3.40E-02

6.32E-05

Cellulose_synthase

Hormone

PGSC0003DMG400014452

966

346

1795

1904

3.34E-02

1.33E-01

Auxin_response_factor 2

Hormone

PGSC0003DMG400028694

1132

330

1330

975

4.05E-02

1.67E-05

Ethylene_receptor_homolog

Flowering

PGSC0003DMG400001453

3296

798

1306

1529

2.91E-02

5.66E-04

RNA-binding_protein

Flowering

PGSC0003DMG400029829

2102

3550

9228

8869

1.07E-02

9.70E-02

Eukaryotic_initiation_factor_3E_subunit

Flowering

PGSC0003DMG400018795

1366

33

79

100

1.22E-02

3.01E-02

Multidrug_resistance_protein_1,_2

Flowering

PGSC0003DMG400014452

966

346

1795

1904

3.34E-02

1.33E-01

Auxin_response_factor_2

Flowering

PGSC0003DMG400024249

782

1654

459

835

7.52E-03

1.02E-08

Gibberellin_20-oxidase-l

Flowering

PGSC0003DMG400032166

567

115

669

718

2.32E-02

5.94E-02

Lysyl-tRNA_synthetase

Flowering

PGSC0003DMG400017035

500

1292

2559

2434

1.76E-02

2.74E-01

RAPTOR1B

Flowering

PGSC0003DMG400002895

44

1252

196

222

2.09E-02

4.14E-02

Sucrose_synthase

Flowering

PGSC0003DMG400004634

222

1178

419

480

3.17E-02

1.40E-02

Sentrin_sumo-specific_protease

Flowering

PGSC0003DMG400008366

206

1140

545

673

3.84E-02

3.83E-03

FI ACA_ribonucleoprotein_complex_subunit

Flowering

PGSC0003DMG400008431

211

641

573

612

7.02E-03

1.00E-01

Protein_arginine_n-methyltransferase_1

RBPs

PGSC0003DMG400007507

1166

443

1297

1422

4.23E-02

1.47E-02

3-5-exoribonuclease_RNA_binding

RBPs

PGSC0003DMG400021249

110

508

1413

1388

6.95E-03

2.93E-01

RNA-binding_protein

RBPs

PGSC0003DMG400022220

293

1083

7037

7840

4.86E-02

2.45E-03

RNA_Binding_Protein_45

RBPs

PGSC0003DMG400023660

79

567

835

814

4.17E-02

2.29E-01

RBP50

Unique and DE genes of petiole-PAC and stem-PAC

Even though petiole- and stem-PAC transcriptomes are similar, there are several transcripts that were expressed uniquely in each. The differentially and uniquely expressed genes indicate the slight difference between petiole phloem and stem phloem. With 10 reads as a mean threshold, approximately 11 k of the genes are expressed in both petiole-PAC and stem-PAC (Table 1, whole genome column, Group 2). There are 1412 and 2710 unique genes expressed in petiole-PAC and stem-PAC, respectively (Table 1, Group 2). Few of these are highly expressed exemplified by the fact that there are only ten petiole-PAC unique genes with greater than 500 reads (Table 3), and only 26 in stem-PAC (Table 4). Several of these highly expressed PAC genes have been functionally characterized in other organisms, such as AP2, an ERF domain-containing transcription factor [37, 38], sucrose synthase [39, 40], and several other DNA- or RNA-binding proteins. The pentatricopeptide repeat-containing protein (PPR) is a RNA-binding protein essential for RNA editing in chloroplasts and mitochondria [41, 42]. PPR proteins help to restore fertility to cytoplasmic male-sterile plants [43] and are involved in organelle biogenesis [44]. Also included in the signaling category, are FRIGIDA, a scaffolding protein involved in flowering, that functions in the formation of a complex that includes both general transcription and chromatin-modifying factors [45] and a jmjC-domain protein. A rice jmjC-domain protein functions in controlling suppression of flowering [46].
Table 3

Genes uniquely expressed in petiole phloem-associated cells

 

Petiole phloem

Stem phloem

P-value

Q-value

Annotation

PGSC0003DMG400008734

661

0

0.000

0.001

AP2_ERF_domain-containing_transcription_factor

PGSC0003DMG400010442

576

1

0.003

0.017

Pentatricopeptide_repeat-containing_protein

PGSC0003DMG400018147

760

5

0.072

0.179

Gene_of_unknown_function

PGSC0003DMG400025480

593

7

0.059

0.163

Receptor_protein_kinase

PGSC0003DMG400030897

674

3

0.016

0.075

Gene_of_unknown_function

PGSC0003DMG400036011

671

4

0.026

0.103

Gene_of_unknown_function

PGSC0003DMG401005729

531

7

0.316

0.331

Cell_wall-associated_kinase

PGSC0003DMG401011335

733

7

0.268

0.311

UDP-glucuronic_acid_decarboxylase_2

PGSC0003DMG401025754

886

9

0.088

0.196

Gene_of_unknown_function

PGSC0003DMG402003286

524

7

0.005

0.033

ACI112

Table 4

Genes uniquely expressed in stem phloem-associated cells

 

Petiole phloem

Stem phloem

P-value

Q-value

Annotation

PGSC0003DMG400001320

2

769

0.006

0.036

Alpha-tubulin

PGSC0003DMG400001999

2

621

0.097

0.206

BZIP_protein

PGSC0003DMG400002009

8

746

0.005

0.032

Hsp20_alpha_crystallin_family_protein

PGSC0003DMG400002303

7

1631

0.003

0.019

Glucan_endo-l,3-beta-glucosidase

PGSC0003DMG400002728

1

581

0.155

0.251

Homeodomain

PGSC0003DMG400006943

6

712

0.242

0.299

Cation_efflux_family_protein

PGSC0003DMG400009942

1

561

0.144

0.245

Gene_of_unknown_function

PGSC0003DMG400011331

6

536

0.118

0.225

Dynein_light_chain

PGSC0003DMG400013186

5

985

0.263

0.309

Ubiquitin_carboxyl-terminal_hydrolase

PGSC0003DMG400015598

2

681

0.059

0.162

Conserved_gene_of_unknown_function

PGSC0003DMG400019310

4

826

0.263

0.309

Jumonji (jmjC)-domain containing_protein

PGSC0003DMG400019353

2

564

0.108

0.217

Conserved_gene_of_unknown_function

PGSC0003DMG400020660

4

660

0.101

0.211

Protein_kinase_domain_containing_protein

PGSC0003DMG400023407

3

715

0.233

0.296

RRM-containing_protein

PGSC0003DMG400026029

1

508

0.006

0.034

MetalloendopepVdase

PGSC0003DMG400026879

3

821

0.080

0.188

Ubiquitin-associated_TS-N_domain-containing protein

PGSC0003DMG400028078

9

547

0.030

0.113

Metalloendopeptidase

PGSC0003DMG400029153

7

807

0.045

0.141

Amino_acid_transporter

PGSC0003DMG400030178

5

817

0.122

0.228

Gene_of_unknown_function

PGSC0003DMG400030396

1

574

0.026

0.105

Conserved_gene_of_unknown_function

PGSC0003DMG400030555

3

537

0.143

0.244

Acetylglucosaminyltransferase

PGSC0003DMG400031046

6

3416

0.006

0.033

Sucrose_synthase

PGSC0003DMG400033575

5

1292

0.186

0.272

Poly(RC)-binding_protein

PGSC0003DMG400034493

4

588

0.043

0.138

FRIGIDA

PGSC0003DMG401023562

1

647

0.108

0.216

Ubiquitin_thiolesterase

PGSC0003DMG403024767

2

557

0.004

0.027

Pectinesterase

Gene ontology categories analyzed for the DE genes were also considered for the uniquely expressed genes in petiole-PAC and stem-PAC (Table 1, Group 2). With 10 reads as a cut-off, the genes uniquely expressed in the stem-PAC are approximately two-fold more in number than the genes uniquely expressed in the petiole-PAC in each category (Table 1, Group 2). Whereas, when considering only the most abundant RNAs (>500 average reads), the number of uniquely expressed genes from both sources is comparable (Table 1, Group 3). These latter abundant transcripts are plausibly important regulators of phloem function or mobile transcripts present in the sieve elements. In summary, there are approximately 1000 genes in these groups (signaling, light, hormone, flowering, transcription factors, RNA-binding) including both unique and common (Table 1, Group 3, red ovals). A complete list of these genes can be found in Additional file 5: Table S4.

Among these genes in the select GO categories of Table 1, many are important regulators of development that may provide insights into the differences between petiole-PAC and stem-PAC. Pseudo-response regulator 5 (PGSC0003DMG400000584) is a well-characterized gene, which has an important role in circadian rhythm [47]. It is classified as a DE petiole-PAC gene, with 1211 reads in the petiole-PAC and only 366 reads in the stem-PAC. BEL33 (PGSC0003DMG400024267) is a flowering-related gene in the TALE (Three Amino Acid Loop Extension) superfamily [48]. Its expression level in stem-PAC is almost twice that of petiole-PAC levels. Gibberellin_receptor GID1 (PGSC0003DMG400028559) is a GA receptor that regulates hormone responses [49]. It is expressed in petiole-PAC with a mean of 1017 reads, five times more than levels in stem-PAC. BRI1 protein is a leucine-rich repeat protein localized to the membrane with an extracellular brassinosteroid receptor domain and intracellular kinase domain [50] and functions in controlling the autonomous flowering pathway [51]. The ccr4-associated protein has mRNA deadenylation activity and is functional in defense [52]. The chromo-domain protein, LHP1, is involved in epigenetic silencing of target genes such as flowering genes. Genetic experiments have shown that LHP1 can affect flowering time and vegetative growth [53].

RNA-binding proteins (RBP) interact with transcripts to mediate numerous aspects of RNA metabolism [54] and function as chaperones that facilitate the long-distance transport of phloem-mobile mRNAs [20]. In the transcriptome of PAC, 364 out of the 464 RBPs in the potato genome were detected (Additional file 6: Table S5). Several of these RBPs have been documented in the literature. The pumpkin ortholog of StPTB1 and StPTB6 (Table 5) was identified as the core protein in a mobile nucleoprotein complex in pumpkin phloem [20]. StPTB1 and StPTB6 play important roles in regulating the movement of the mobile RNA, StBEL5 in potato [55]. IF2 is the potato ortholog of Nova, a KH domain RBP, that binds to StPTB1 and StPTB6 [56]. A PTB7-like protein was also identified in pumpkin phloem and its orthologs in Arabidopsis have been implicated in alternative splicing [2, 57]. Several of the RBPs identified using the 3′ UTR of StBEL5 as bait in the yeast three-hybrid system [58] were also detected in the petiole- and stem-PAC transcriptomes (Table 5). These include sucrose synthase, eIF5A, and a glycine–rich RBP. Four pumilio proteins containing a Puf domain that interacts with 3′ UTRs in target RNAs were detected. All four were relatively abundant in both petiole and stem profiles. Pumilio has only recently been discovered in plants but is widespread in numerous species and functions in diverse aspects of mRNA metabolism that regulate development and defend against viruses [5961].
Table 5

Abundance of RNA-binding proteins in phloem-associated cells

Gene

Gene ID

Petiole phloem

Stem phloem

Identified in

Citation

StPTBl

PGSC0003DMG400018824

234

373

Ortholog of CmRBP50

Ham et al., [20]; Butler et al. [35]

StPTB6

PGSC0003DMG400023660

79

567

Ortholog of CmRBP50

Ham et al., [20]; Butler et al., [35]

StPTB7

PGSC0003DMG400001427

140

188

In potato PTB family

Rühl et al., [57]

StPTB7-l

PGSC0003DMG400009106

28

43

In potato PTB family

Rühl et al., [57]

StPTB7-2

PGSC0003DMG400002353

18

16

In potato PTB family

Rühl et al., [57]

StPTB7-3

PGSC0003DMG400019613

101

391

In potato PTB family

Rühl et al., [57]

RBP1

PGSC0003DMG402017409

652

1005

pumpkin phloem proteome

Lin et al., [2]

GRP7

PGSC0003DMG400000708

99,744

161,520

pumpkin phloem proteome

Lin et al., [2]

RBP-45

PGSC0003DMG400011290

1217

724

pumpkin phloem proteome

Lin et al., [2]

RBP6

PGSC0003DMG400012601

2747

2147

pumpkin phloem proteome

Lin et al., [2]

Albal

PGSC0003DMG400020480

1

64

pumpkin phloem proteome

Lin et al., [2]

Alba2

PGSC0003DMG400020460

0

43

pumpkin phloem proteome

Lin et al., [2]

Pumiliol

PGSC0003DMG400002143

4748

5411

Leaf development

Huang et al., [61]

Pumilio2

PGSC0003DMG400006350

344

669

Potato PAC

Abbasi et al., [59]

Pumilio3

PGSC0003D MG400009166

834

343

Potato PAC

Abbasi et al., [59]

Pumilio4

PGSC0003DMG400030327

751

1109

Potato PAC

Abbasi et al., [59]

IF2 (Nova)

PGSC0003DMG400023482

227

329

pumpkin phloem Y2H with PTB1 and PTB6

Lin et al., [2]; Shah et al., [56]

IF1 (RRM protein)

PGSC0003DMG400008877

600

1353

Y2H with PTB1 and PTB6

Shah et al., [56]

Sucrose svnthase-4

PGSC0003DMG400013546

4219

7886

Y3H with StBEL5 3′ UTR

Cho et al., [58]

GR-RBP3

PGSC0003DMG400028111

345

355

Y3H with StBEL5 3′ UTR

Cho et al., [58]

LSH10

PGSC0003DMG400020442

44

22

Y3H with StBEL5 3′ UTR

Cho et al., [58]

elF5A

PGSC0003DMG400011137

1077

1216

Y3H with StBEL5 3′ UTR

Cho et al., [58]

B5RBP5

PGSC0003DMG400031406

352

527

Y3H with StBEL5 3′ UTR

Cho et al., [58]

Zinc finger

PGSC0003D MG400027176

4424

1879

Y3H with StBEL5 3′ UTR

Cho et al., [58]

B5RBP7

PGSC0003DMG400017156

4323

5245

Y3H with StBEL5 3′ UTR

Cho et al., [58]

To validate expression patterns of select RBPs without the bias of amplification or the imbedding techniques inherent in the LCM protocol, a sample of RNA profiles from the Potato Genome Sequencing Consortium [62] was assembled in eight different organs for 26 RBPs (Fig. 3). These were selected on the basis of their relative abundance in petiole-PACs, their binding affinity for the 3′ UTR of the mobile RNA, StBEL5 [58], or the presence of their protein orthologs in pumpkin phloem sap [2]. Consistent with their abundance in petiole-PAC, most of these proteins exhibited significant levels of transcripts in petioles (Fig. 3, red arrows). Among the samples, three RNA helicases, one PTB protein, and two glycine-rich RBPs were profiled. Plant helicases have a known function in regulating the size exclusion limit of plasmodesmata [63]. Several RBPs exhibited spikes in transcript levels in specific organs (Fig. 3; Additional file 7: Table S6). RBP6, RBP RZ-1, and glycine-rich protein 2 were relatively abundant in young tubers. RBP-45 and one of the Dead-box RNA helicases exhibited select relative abundance of transcripts in stolons. Two of the “RNA-binding proteins” and the FUS-interacting ser/arg-rich protein-1 all spiked in roots. These observed abundance levels may reflect a putative organ-specific function for select RBPs.
Fig. 3

Expression profiles of select RNA-binding proteins mined from RNA-seq data from the Potato Genome Database from the Tuberosum RH89-039-16 haplotype [62]. RNA profiles from eight organs are presented and medium (a) and high (b) abundance values are shown in FPKMs (fragments per kb per million mapped reads). Selection of these RNAs was based on their relative abundance in petiole-PACs, their binding affinity for the 3′ UTR of the mobile RNA, StBEL5* [58], or the presence of their protein orthologs in pumpkin phloem sap** [2]. Red arrows designate petiole samples. Accession numbers for these genes are listed in Additional file 7: Table S6

Abundant transcripts in phloem-associated cells

Transcriptome profiling identified a plethora of genes that are highly expressed in petiole PAC or stem PAC. The highest average total reads for one specific gene were 154,207 in petiole PAC and 274,582 in stem PAC. In the petiole-PAC library, 1209 genes had >1000 reads and 2626 genes had >500. In the stem-PAC library, 1288 genes had >1000 reads and 2822 genes had >500. There are 3593 genes with >500 reads in either petiole-phloem or stem-phloem associated cells (Additional file 8: Table S7). To compare the genes highly expressed in PAC with other genes in the whole genome, these 3593 genes were regarded as genes with abundant transcripts and were analyzed for attributes based on gene annotations.

Approximately 2971 GO terms were involved with the abundant transcripts in PAC. The GO categories identified for each gene were made comparable by converting each GO term to the same level in the hierarchy to permit clustering into GOslim categories. This was done with goslimviewer using AgBase software. Twenty-five cellular components, 44 biological processes and 26 molecular functions were applied (Fig. 4). The most abundant GO categories represented were “binding” and “nucleic acid binding” (Fig. 4). By comparing to the whole genome with GOseq [64], the gene ontology of the abundant transcripts revealed 511 categories over-represented with adjusted p-values smaller than 0.05. In the 510 over-represented GO categories there are 101 molecular functions, 73 cellular components and 336 biological processes. Many binding-related functions were verified as over-represented categories with adjusted p-values smaller than 0.05. Ion binding functions, such as zinc ion binding, copper ion binding, cobalt ion binding and calcium ion binding, were all over-represented (Fig. 5a). RNA-binding and protein-binding functions were also over-represented in active PAC genes. These binding functions likely contribute to facilitating the transport processes of phloem. The PAC-abundant transcripts are also involved with numerous important biological processes, including both response and developmental activities (Fig. 5b-c). The top 32 over-represented molecular function and biological processes related to signaling included responses to both light quality and quantity (Fig. 5c). These signals are commonly perceived in the leaf, and phloem in the leaf veins and petiole serve as the conduit to deliver these signals to other organs. There are also several flowering-related GO categories over-represented in the abundant transcripts, including photoperiodism, flowering, ovule development, regulation of flower development, and flower morphogenesis (Fig. 5c). Among the 175 GO categories related to signaling, as listed in Table 1, 34 of them are over-represented in the PAC-abundant transcripts (all listed in Fig. 5c).
Fig. 4

Distribution of molecular functions in the abundant transcripts in phloem associated cells. The reads of all the genes identified in petiole-PAC or stem-PAC were normalized with upper quantile normalization. A mean value is calculated from the three replicates. Genes with more than 500 reads in the mean value of either petiole-PAC and stem-PAC were regarded as abundantly expressed genes. GO terms of the whole genome were analyzed with Blast2GO. The GO categories identified for each gene were made comparable by converting each GO term to the same level in the hierarchy to permit clustering into GOslim categories. This was done with goslimviewer using AgBase software [96] (http://agbase.msstate.edu/cgi-bin/tools/goslimviewer_select.pl)

Fig. 5

Over-represented molecular function and biological processes in phloem abundant transcripts. GO terms involved with DE genes between petiole-PAC and stem-PAC were analyzed with GOseq [64] to compare their enrichment in DE genes relative to the whole genome. The p-value is adjusted with BH method [97]. The over-represented genes were defined with adjusted p-value smaller than 0.05. The ratio of each GO term is calculated by dividing the number of genes involved with each GO term with number of genes in the whole group. a includes the top 20 over-represented GO terms of molecular function and (b) includes the top 20 over-represented GO terms of biological processes. c shows the GO terms related to transcription factor, flowering, light, signaling and hormone that are over-represented

The effect of photoperiod on the transcriptome of petioles

Day length regulates numerous aspects of plant development and is especially important in potato for controlling tuber formation. The perception of length of daylight/darkness generates leaf-derived signals that are traansported throughout the whole plant through petiole/stem vascular connections [9, 13, 14]. To identify genes that are regulated in petioles in response to photoperiod, we sequenced RNA samples from petioles of the photoperiod-responsive species Solanum tuberosum ssp. andigena under long-day (LD) and SD photoperiods using RNA-seq. Four replicate samples for each were isolated from the petiole tissues harvested from plants grown under long- and short-day conditions. The reads were mapped to the potato genome with GSNAP, and the number of concordant unique reads was counted for each gene using HT-seq (Additional file 9: Table S8). The number of reads contained in each library was greater than 1 × 107 and approximately 94 % of the paired reads were mapped to the genome in a concordant and unique manner. Of the 39,028 genes contained in the potato, approximately 25 k genes were detected in the whole petiole samples (LD Petiole and SD Petiole) with at least one read aligning to the gene (Additional file 9: Table S8). Representing only a few cell types from the petiole and stem organs, RNA-seq results of PAC scored significantly fewer genes than the whole petiole sample (Additional file 1: Table S1). The genes expressed in phloem are very likely detected in the whole petiole samples depending on the depth of the petiole profile. Beyond PAC, there are many other cell types in the petiole (e.g., collenchyma, sclerenchyma, palisade parenchyma, and epidermis), so a proportion of genes will likely only be detected in the whole petiole. In theory, the genes with higher reads in PAC represent genes with a significant putative function in the phloem. Of course, this set of genes will also be detected in the whole petiole samples, albeit at a lower proportion of reads (Additional file 3: Table S2).

Reads of the eight libraries were normalized using the upper quantile normalization approach. A generalized linear model was applied to the LD Petiole and SD Petiole results with the R package “QuasiSeq” to analyze the photoperiod effect. All other conditions were kept consistent and the libraries were sequenced with multiplexing tag in the same lane, so photoperiod effect is the only fixed effect that was considered (Additional file 10: Figure S2). Eleven-thousand, nine-hundred, fifty-seven genes were identified as significantly DE with q-values less than 0.05 (Additional file 11: Table S9). Means of the normalized reads of the four replicate libraries for both long- and short-day treatments were used to indicate their measured level. Among the 20,564 genes with at least 10 reads in either LD or SD petiole, 517 of them are uniquely expressed in LD, and only 388 of them are uniquely expressed in SD. Most of these uniquely expressed genes exhibited low abundance read values. The most abundant transcript uniquely expressed in LD has only 558 reads (PGSC0003DMG400026590) whereas the most abundant unique transcript under SD conditions has only 208 reads (PGSC0003DMG400016462). Among the 11,957 DE genes, 5555 of them are up-regulated under LD, and 6402 of them under SD. Twelve-hundred and eight of the DE genes activated by LD have a log2-fold change more than 1, and 248 genes out of the 1208 have reads more than 500 under LD (Additional file 12: Table S10). Eight-hundred and twenty of the DE genes activated by SD have a log2-fold change more than 1, and 128 genes out of the 820 have reads more than 500 under SD (Additional file 13: Table S11). Transcripts that are regulated by photoperiod and are relatively abundant (>380 reads) in petiole-PAC may be indicative of genes involved in signaling or transport mediated by day length. Included in this list are genes encoding for the Agamous-like MADS-box protein/AGL8 ortholog, a circadian clock-associated FKF1 protein, Pseudo-response regulator 5, the AP2 ERF-domain protein, an ethylene receptor, a NAC-domain protein, and a nodulin MtN3 family protein (Additional file 12: Table S10 and Additional file 13: Table S11). As an example, the AGL8 ortholog of potato is induced by the StFT-like tuberization signal, SP6A [14], and is involved in controlling meristem and tuber development by regulating cytokinin levels [65]. Several notable DE photoperiod genes were selected to verify their relative expression levels with qRT-PCR (Fig. 6a). Four were up-regulated by SD (Fig. 6b) and four by LD (Fig. 6c). All eight exhibited expression patterns consistent with their comparable RNA-seq data.
Fig. 6

Select genes that are differentially expressed in petioles of potato in response to photoperiod (a). Mean reads from RNA-seq data are presented for both petiole- and stem-PAC and for LD/SD petioles. Real time RT-PCR was used to verify the RNA-seq results for SD-induced (b) or LD-induced (c) genes. Each gene in panels b and c is designated by its 5-digit PGSC identifier (in bold, a). Relative levels of their transcripts were quantified using total RNA extracted from petiole samples harvested from plants grown under long (open bars) or short (shaded bars) days. Short-day plants were harvested after 10day of SD conditions (8 h light, 16 h dark). Quantitative real-time RT-PCR with gene-specific primers was used to calculate the relative amounts of RNA for each gene. The expression of each gene was calculated as the 2-ΔCt value and normalized to the endogenous reference gene, StAct8. Standard errors of the means of three biological replicates are shown with 1 and 3 asterisks indicating significant differences (p < 0.05, p < 0.001, respectively) using a Student’s t test

Gene ontology of photoperiod-regulated genes of the petiole

To visualize functional relationships in this diverse expression profile, the 11,957 DE genes were also analyzed for their gene ontology categories. Four-thousand, four-hundred, and twenty-nine GO terms were applied to the 11,957 photoperiod DE genes, including 6056 of cellular component, 34,632 of biological process and 29,235 of molecular function. GO distribution of the photoperiod DE genes was analyzed with GOseq to identify the over-represented GO groups. With familywise adjusted p-value <0.05, GO terms for 64 cellular components, 136 molecular functions, and 369 biological processes were significantly enriched in the photoperiod DE genes (Fig. 7; Additional file 14: Figure S3 and Additional file 15: Figure S4). As expected, “circadian rhythm” is identified as an over-represented GO category as well as several light-related GO terms (Fig. 7). Among the molecular functions, binding is the most over-represented function, including binding to ATP, protein, nucleotide, DNA, RNA, and several kinds of ions (Additional file 14: Figure S3).
Fig. 7

Over-represented GO terms related to light signaling in DE photoperiod genes. GO terms involved with DE genes between long- and short-day treatments were analyzed with GOseq [64] to compare their enrichment in DE genes relative to the whole genome. The p-value was adjusted with BH method [97]. The over-represented genes were defined with adjusted p-value smaller than 0.05. The ratio of each GO term is calculated by comparing the number of genes involved with each GO term with number of genes in the whole group

RNA-binding motifs

Mobility of mRNA, stability and control of translation are facilitated by RNA-binding proteins associated with them. RBPs commonly bind to conserved elements in the 3′ un-translated region (UTR) of the RNA. To assess the frequency of select RBP motifs in potato transcripts, downstream sequence (DSS) from the stop codon was screened for the presence of RBP motifs. As a reference, in Arabidopsis, the average length of the 3′ UTR is 248 nt [66]. Three known RBP target elements were searched, including those for polypyrimidine tract-binding proteins (PTB), Pumilio and Nova, a KH-domain protein [67] (Additional file 16: Figure S5A-C). Pumilio was selected because of its relative abundance in PAC (Table 5), its functional relevance, and its widespread role in RNA metabolism [68]. PTB and Nova were selected because of their prominence in binding to RNAs and because both were detected in phloem sap of cucumber suggesting they are both phloem mobile [2]. The Pumilio binding motif has been confirmed as UGUAu/c/aAUA where the 5th nucleotide can be U, C, or A [69], whereas Nova’s is modeled as u/c/aCAUUUCAc/u [67]. PTB proteins bind to RNA at four RNA recognition motifs (RRM). Each RRM can interact with a cytosine/uracil (CU) motif ranging from 3 to 6 nt. In our search, the PTB motif was defined as a cluster of four CU runs each, at least, 4 nt in length within the designated DSS. Biochemical analysis of interactions of target RNA to the binding pockets of PTB protein domains demonstrated that binding to PTBs is not sequence-specific and that many RNA fragments readily bind to them [70].

Using the MEME suite [71] and BEDTools [72], Pumilio, Nova, and PTB binding motifs were initially searched across the genome and extracted through 1000 nt of DSS (Additional file 16: Figure S5A-C). Because of their frequent occurrences, all three motifs were again searched through either 500 (Pumilio and Nova) or 200 (PTB) nt of DSS (Table 6). Any transcripts that contained at least four CU runs within this 200-nt DSS were identified as potential PTB targets. Forty-six hundred RNAs were identified with a Pumilio binding motif, 3000 RNAs with a Nova binding motif, and more than 3000 RNAs contain the PTB motif (Table 6, Group 1). Ham et al. [20] demonstrated that the pumpkin PTB protein, RBP50, binds specifically to UUCUCUCUccuUCUU sequences present within a subclass of phloem-mobile, polyadenylated transcripts. On this basis and to ensure more exclusiveness, we screened DSSs of the 31,742 PTB RNA pool for this motif. From this screen, 422 RNAs were identified that contained the RBP50 motif. Included in this list were RNAs encoding a Gag-pol polyprotein (HIV-related), an integrase core domain-containing protein (HIV-related), ethylene response factors, a SET-domain protein, a LOB-domain protein, a tuber-specific element-binding protein, and numerous other TFs, signaling and receptor-like proteins.
Table 6

Transcripts with RNA-binding motifs in the 3′ UTR (500 or 200 bp downstream of stop codon)

 

Pumilio (500 bp)

Nova (500 bp)

PTB (200 bp)

Group 1

 Transcripts in whole genome (39,028)

4629

3042

31,742

 Transcripts with more than 10 reads in phloem-associated tissue (15,167)

2142

1318

13,320

 Transcripts with more than 500 reads in phloem-associated tissue (3593)

529

290

3223

Group 2

 PAC abundant & Differentially expressed genes under photoperiod change (2166)

338

168

1943

Group 3

 TFs (sequence-specific DNA binding transcription factor activity) (1090)

190

88

859

 TF transcripts with more than 10 reads in PAC (525)

103

39

442

 TF transcripts abundant in PAC (163)

28

6

130

After examining the frequency of distribution of these motifs in DSS, only the Pumilio motif demonstrated any significant enrichment. This enrichment occurred in the first 200 nt of DSS. In contrast, the motifs for PTB and Nova were randomly distributed across all DSS examined (Additional file 16: Figure S5A-C). Many of the DSSs analyzed here contained multiple binding motifs. Among the 4629 RNAs containing a Pumilio binding motif, there are 383 with two Pumilio binding motifs, 43 with three, 6 with four and 1 with seven. Among the 3042 RNAs containing multiple Nova binding motifs, there are 176 with two motifs, 10 with three, and 1 with four motifs. There are also different motifs existing in the same DSS (Additional file 16: Figure S5D). Three hundred and twenty DSSs screened contained all three types of RBP motifs. Approximately, 6000 transcripts contained two different RBP binding motifs of Pumilio, Nova or PTB. Unique among the three motifs we searched, Pumilio motif-targeted transcripts were over-represented in 13 gene ontology categories (Additional file 17: Figure S6). Included among these categories were “sequence-specific DNA binding transcription factor activity”, “protein autoubiquitination”, “DNA-dependent regulation of transcription” and “DNA-dependent negative regulation of transcription”. All these functions and biological processes are important in regulating their targets and downstream genes. By its binding to the transcripts of regulatory genes, Pumilio protein indirectly regulates the activity of a wide range of genes.

Accumulation of RBP targets in PAC

The RBP targets abundantly expressed in PAC are of especial interest. Present in sieve elements, phloem-mobile transcripts associated with RBPs are good candidates for long-distance signals. Among the PAC-abundant transcripts (>500 in petiole-PAC or stem-PAC), 529 contain the Pumilio binding motif, 290 contain the Nova binding motif, and 3223 contain the PTB binding motif (Table 6, Group 1). When considering the photoperiod effect, these numbers are reduced even more at 338, 168, and 1943, respectively (Table 6, Group 2). As an over-represented GO group in Pumilio-targeted transcripts, the 1090 “sequence-specific DNA binding transcription factor activity” related genes in the potato genome (Additional file 17: Figure S6) were also screened for Nova and PTB binding motifs. Among the transcripts of transcription factors in PAC (>10 reads in petiole-PAC or stem-PAC), 103 of them are Pumilio targets, 39 are Nova targets and 442 are PTB targets (red text, Table 6, Group 3; Additional file 18: Table S12). Only 28, 6, and 130 of these transcripts were abundant in PAC (>500 reads), respectively, with Pumilio, Nova and PTB binding motifs.

Several TF families exhibited enrichment of these RBP binding motifs in their DSSs (Table 7). Auxin responsive factors (ARF) and AUX/IAA are two different components in auxin-mediated transcription regulation, as transcription factor and transcriptional repressors, respectively [73]. AUX/IAA transcripts have been reported to be phloem mobile into roots [12] and eighteen RNAs in this class contain at least one RBP binding motif. One notable RNA is Auxin response factor 2 (PGSC0003DMG400014179). This RNA accumulates to high levels in both petiole-PAC and stem-PAC and both PTB and Pumilio motifs are present in its DSS. Long-distance movement of transcripts encoding both StBEL1 and StKNOX type TFs has also been previously reported [9, 21]. Only two of the 13 StBEL RNAs, StBEL22 and −30, contained no RNA-binding motifs for the three RBPs we searched. DSS of STH1 contains all three RNA-binding motifs. All six KN1-like RNAs contain PTB motifs and three of the six are relatively abundant in PACs (Table 7). Other abundant PAC TFs containing RNA motifs include members of the NAC-domain [74] and WRKY [75] families. Two NAC domain RNAs, Nam 9, and two WRKY transcription factors contain both PTB and Pumilio binding motifs in their DSS (Table 7).
Table 7

Transcription factors with RNA-binding protein motifs in their downstream sequences

Gene ID

Motif

Annotation

Pet phloem

Stem phloem

LD. mean

SD. mean

PGSC0003DMG400002392

PTB

  

Auxin_response_factor_7

309

212

954

1209

PGSC0003DMG400003771

PTB

  

Auxin_response_factor_5

11

149

282

437

PGSC0003DMG400008065

PTB

  

Auxin_response_factor_4

910

835

2160

2426

PGSC0003DMG400009773

PTB

  

Auxin_response_factor_19

540

1364

2414

2546

PGSC0003DMG400014179

PTB

PUM

 

Auxin_response_factor_2

1805

2124

4654

5835

PGSC0003DMG400014452

  

NOVA

Auxin_response_factor_2

966

346

1795

1904

PGSC0003DMG400015919

PTB

  

ARF8

826

600

4562

4915

PGSC0003DMG400020711

 

PUM

 

Auxin_response_factor_1

750

822

3921

3179

PGSC0003DMG400023345

PTB

  

ARF_domain_class_transcription_factor

500

206

806

637

PGSC0003DMG401018664

PTB

  

Auxin_response_factor_8-1

557

508

4626

3659

PGSC0003DMG400000118

PTB

  

StlAA15 (AtARF9)

2638

2838

2945

3706

PGSC0003DMG400002608

  

NOVA

StlAA14 (AtlAA18/28)

926

1325

2393

693

PGSC0003DMG400005327

PTB

  

StlAAlO (AtlAA16)

710

549

958

1483

PGSC0003DMG400006093

PTB

  

StlAA24 (AtlAA14)

413

178

6503

11,614

PGSC0003DMG400013445

PTB

PUM

 

StlAA12 (AtlAA3)

0

61

21

20

PGSC0003DMG400020139

PTB

PUM

 

StlAA2 (AtlAAl)

11

23

382

469

PGSC0003DMG400029339

PTB

  

StlAA5 (AHAA13)

4

125

36

46

PGSC0003DMG402019457

PTB

  

Auxin_indole-3-acetic_acid_3(AtlAA16)

534

537

6031

5908

PGSC0003DMG400019635

PTB

  

BEL11

92

85

4043

5214

PGSC0003DMG400010086

PTB

  

BEL13

1

162

520

389

PGSC0003DMG400012329

PTB

  

BEL14

0

0

84

83

PGSC0003DMG400021323

PTB

  

BEL29

1282

2591

4788

4997

PGSC0003DMG400003750

PTB

  

BEL31

16

9

382

336

PGSC0003DMG400024267

PTB

PUM

 

BEL33

464

812

1072

1100

PGSC0003DMG400008057

PTB

PUM

 

BEL34

199

66

218

399

PGSC0003DMG400005930

PTB

  

BEL5

2089

1234

3602

3375

PGSC0003DMG400003751

 

PUM

 

BEL32

10

47

958

680

PGSC0003DMG400019142

 

PUM

 

BEL35

301

453

528

635

PGSC0003DMG400029946

 

PUM

 

BEL6

5

60

204

282

PGSC0003DMG400030961

   

BEL30

9

23

770

769

PGSC0003DMG400022011

   

BEL22

0

84

57

50

PGSC0003DMG400007887

PTB

PUM

 

Homeobox_protein_knotted-l-like_LET12

12

13

84

53

PGSC0003DMG400030737

PTB

  

Homeobox_protein_knotted-l-like_LET12

1132

2984

3147

3270

PGSC0003DMG400004953

PTB

  

STH20

607

726

186

226

PGSC0003DMG400013493

PTB

  

POTH1

267

24

77

98

PGSC0003DMG400016711

PTB

  

STH15 (STM)

4341

3154

192

185

PGSC0003DMG400002769

PTB

PUM

NOVA

STH1

55

61

567

499

PGSC0003DMG400011891

PTB

PUM

 

NAC_domain_protein

172

25

1368

624

PGSC0003DMG400016896

PTB

  

NAC_domain_protein

117

266

125

93

PGSC0003DMG400019615

PTB

  

NAC_domain_protein

112

30

192

179

PGSC0003DMG400032555

PTB

  

NAC_domain_protein

128

4

112

91

PGSC0003DMG400018435

PTB

PUM

 

NAC_domain_protein_NAC2

202

104

215

99

PGSC0003DMG400017567

PTB

  

Nam

461

290

1067

663

PGSC0003DMG400028662

PTB

  

Nam 2

1248

1999

3026

2292

PGSC0003DMG400031072

PTB

  

Nam 4

1998

1083

1034

803

PGSC0003DMG400031149

PTB

  

Nam 7

347

442

809

685

PGSC0003DMG401023373

PTB

  

Nam 8

47

101

502

471

PGSC0003DMG400031071

PTB

PUM

 

Nam 9

925

1492

1621

1761

PGSC0003DMG402023373

PTB

  

Nam 9

395

216

1428

1310

PGSC0003DMG400000064

PTB

  

WRKY_transcription_factor_23

67

37

98

134

PGSC0003DMG400000211

PTB

  

WRKY_transcription_factor

520

126

236

164

PGSC0003DMG400001434

PTB

  

WRKY_transcription_factor-c

9

38

224

166

PGSC0003DMG400005329

PTB

PUM

 

WRKY_transcription_factor

1579

946

976

918

PGSC0003DMG400005836

PTB

  

WRKY_transcription_factor-30

42

0

13

10

PGSC0003DMG400009014

 

PUM

 

WRKY_transcription_factor_lle-1

157

50

1416

1532

PGSC0003DMG400009530

PTB

  

WRKY_transcription_factor_3

974

713

580

1000

PGSC0003DMG400015076

PTB

PUM

 

WRKY_transcription_factor

204

44

184

146

PGSC0003DMG400020432

PTB

  

WRKY_transcription_factor_5

1

82

98

76

PGSC0003DMG400022063

PTB

  

WRKY1a_transcription_factor

17

0

38

43

PGSC0003DMG400022143

PTB

  

WRKY_DNA-binding_protein

1702

1599

2386

1988

PGSC0003DMG400023196

PTB

  

WRKY_transcription_factor

27

28

1

1

PGSC0003DMG400028520

PTB

  

WRKY_transcription_factor_1

3534

736

774

559

PGSC0003DMG401031196

PTB

  

WRKY_transcription_factor_16

571

255

458

186

PGSC0003DMG401033880

PTB

  

WRKY_transcription_factor_27

36

79

326

360

Discussion

Phloem RNA derived from laser capture microdissection

Compared to a genomic study, transcriptome analysis is more informative as it provides a snapshot of processes of physiology and development. RNA levels can be affected by three factors, the rates of transcription, degradation and processes of movement. For phloem-associated cells, the dynamics of transcript levels are even more important, since phloem is the conduit for allocation of photosynthate. In addition to the transcripts that maintain the metabolism and function of phloem, there is also a unique set of non-cell-autonomous mRNAs moving through the phloem as potential long-distance signals. Because of the limitations inherent in the harvest of potato phloem sap, isolation of phloem cells can be readily accomplished by using the well-developed technique of LCM. Early applications of LCM to extract RNA from phloem cells lacked depth and were inefficient. LCM of rice phloem cells yielded only 413 clones that exhibited a high level of redundancy [26]. Refinement of the technique coupled with high-resolution next generation sequencing technology has facilitated expression analysis of select target cells characterized by a very high level of resolution and reproducibility.

In this study, we sequenced the transcriptome of petiole-PAC and stem-PAC using RNA-seq. The raw output is approximately 107 reads for each sample. Out of the approximately 40 k genes in the potato genome, roughly 15 k genes were expressed in PAC of petiole and stem. Our numbers are comparable to the 14,242 and 13,775 active genes identified in the vascular bundles of cucumber and watermelon, respectively [36]. Through statistical analysis, petiole- and stem-PAC exhibited very similar transcriptomes with just slight differences. The genes DE in common between them and the unique genes in each were associated with important GO categories in both signaling and developmental regulation. Approximately 50 DE genes were grouped into signaling-related GO categories, including light, hormone, and flowering related categories. GO categories for binding functions were proportionately over-represented for transcripts abundant in PAC, including both DNA and RNA binding. A propensity for binding and signaling categories for genes expressed in PAC would reflect the dynamic functions of the phloem as a conduit for transporting sucrose and a range of signaling molecules [76].

Previous expression profiles of phloem

Previous work has established the foundation for RNA profiling of phloem utilizing both LMPC-derived cells and sap harvested from Arabidopsis and melon [22, 23]. Transcripts present in sieve elements were identified from melon phloem sap, which readily bleeds from stem cuts [22]. In this study, 1830 unique ESTs were sequenced and mapped to 986 unique transcripts. Using gene functional analysis, 15 % of these genes encoded proteins related to signal transduction. Using these ESTs as a query, 124 potato orthologs were identified from the potato genome. One-hundred and four of them were expressed in either petiole- or stem-PAC. Unfortunately, the profile of the phloem ESTs in melon lacked much depth and expression levels were not verified quantitatively. Using RNA-seq, approximately 104-fold more fragments can be generated and sequenced compared to the EST sequencing approach. This enhanced resolution provides more quantitative sequence information and more insights into the function of the profiled RNAs.

The study on Arabidopsis phloem compared the profiles of LMPC-derived phloem tissue and leaf phloem exudate and in this way, provided a hint of the identity of mobile transcripts present in the phloem [23]. Approximately 2400 transcripts were identified in the phloem exudate by microarray, and 90 of them were categorized as functional in signaling pathways. Seventy-six genes in the potato genome were identified as orthologs of these 90 putative mobile transcripts. Seventy of the 76 genes were expressed (>10 reads) in either the petiole- or stem-phloem libraries (Additional file 19: Table S13). Twenty-eight exhibited more than 1000 reads in the petiole-PAC library. These orthologs, including 14-3-3 proteins, MAP kinases, light-related proteins (e.g., one AUX/IAA RNA), and calcium-responsive signals (Additional file 19: Table S13), represent potential mobile mRNAs in potato. The comparison of profiles in the Deeken et al. study [23] is invaluable but because most plants do not readily yield phloem sap, our current approach using LCM technology coupled with RNA-seq exhibits numerous advantages. It is consistent with previous methods but provides wider applicability, cell specificity, and excellent in-depth sequence resolution. On the downside, the LCM protocol is labor-intensive, may yield small amounts of RNA, and opens the possibility of harvesting cells outside the target tissues. A major advantage of using sap over PACs for examining phloem signaling is the enhanced specificity provided by phloem sap analysis. A PAC profile provides greater overall coverage but less specificity for signal transcripts that move through the sieve element system.

Accumulation patterns for established phloem-mobile mRNAs

RNAs concentrated in PAC can be specifically located in the sieve element, companion cells or parenchyma cells. Depending on stability and transport dynamics, mobile mRNAs may be concentrated in sieve elements. Through RNA-seq, thousands of abundant phloem transcripts can be profiled, but to verify their mobility requires heterografting experiments with different but related species [6] or RNA movement assays [77]. Another option is to generate stably transformed plants that express the test RNA with a non-plant sequence tag and heterograft with wild type plants [9]. All three approaches are labor-intensive and time consuming. The in silico analysis approach for identifying conserved motifs in 3′ UTRs presented in this study (Tables 6 and 7) has the clear potential to more efficiently predict candidate transcripts for long-distance mobility.

Non-cell-autonomous mRNAs may move into the sieve element system through plasmodesmata connecting companion cells to sieve elements [78]. In this model, any RNAs from the leaf, transported long distance, may be detected in both petiole- and stem-PAC. As discussed previously, there are hundreds of full-length mRNAs present in phloem sap but only a few of these have been confirmed to move and even fewer are associated with a phenotype [15]. This short list includes LeT6 in tomato, GAI in pumpkin, tomato, and Arabidopsis, IAA18/28 in Arabidopsis, and POTH1 and StBEL5 of potato. The potato orthologs of these mobile RNAs along with StBEL5 and POTH1 are detected in the phloem-associated cells of both petiole and stem in relatively abundant levels (Table 8). Because there are reports of the transport of FT mRNA [11, 79], the FT/SP6A orthologs of potato are also included. The complete absence of any accumulation of transcripts for StFT genes suggests that these RNAs are not phloem-mobile. Evidence indicates that SP6A is translated in leaves and moves through the phloem to underground stolons in protein form [14]. STH15 and StBEL5 exhibited the greatest levels of accumulation in PAC (Table 8). Of course, abundance levels of any specific RNA would be determined by the rate of transcription, the stability of the RNA, and the degree of its mobilization.
Table 8

Accumulation of known mobile mRNAs in stem and petiole PACs

Annotation

Gene ID

Petiole phloem

Stem phloem

Function

Citation

STH15 (STM, LeT6)

PGSC0003DMG400016711

4341

3154

Leaf morphology

Kim et al., [10]; Ham et al., [20]

DELLA protein GAI

PGSC0003DMG400015692

531

422

Leaf morphology

Haywood et al., [8]

StBEL5

PGSC0003DMG400005930

2089

1234

Tuber and root growth

Banerjee et al., [9]; Lin et al., [19]

IAA18/28

PGSC0003DMG400002608

926

1325

Root growth

Notaguchi et al., [12]

POTH1

PGSC0003DMG400013493

267

24

Vegetative growth

Mahajan et al., [21]

FT

PGSC0003DMG400016179

0

0

Flowering

Li et al., [11]

SP6A

PGSC0003DMG400023365

0

0

Tuberization

Navarro et al., [14]

GA INSENSITIVE (GAI) is exceptional in that long-distance movement of its mRNA has been established in several plant species including cucumber, tomato, pumpkin [8, 20], apple [80], and Arabidopsis [81]. It was the first mobile RNA identified and CU-rich sequences in its transcript facilitate binding to CmRBP50, a PTB protein of pumpkin. Accumulation of AtGAI across a graft union can affect leaf architecture [8]. IAA18/28 was verified to cross graft unions and move into root tips to regulate root architecture [12]. STH15 is the ortholog of LeT6 of tomato and STM of Arabidopsis. Mobility assays of LeT6 confirmed upward movement of is transcript associated with a leaf phenotype in tomato [10]. Both POTH1 and StBEL5 have been associated with tuber development [82, 83]. Movement of StBEL5 is induced by short days and regulated by its UTRs. The UTRs of POTH1, StBEL5, and StGAI interacted with a potato PTB protein [21]. Because several of these mobile RNAs interact with the same RBP, it is conceivable that multiple RNAs are transported in the same RNP complex. For example, the mobile RNA/RBP50 complex of pumpkin contained six RNAs including CmGAI and CmSTM. All six of these RNAs contained CU-rich PTB motifs. These CU-rich motifs were also observed in the UTRs of StBEL5, POTH1, StGAI, and STH15.

The role of RNA-binding proteins

RBPs mediate numerous aspects of RNA metabolism including mRNA capping, rate of degradation, translation, localization and transport. For long-distance mobilization of mRNAs, RBPs associated with them are especially important in stabilizing and localizing the mRNAs, while repressing translation during the process. Our analysis revealed numerous transcripts encoding RBPs in both petiole- and stem-PAC (Additional file 6: Table S5) and it is very likely that a subgroup of these are functional in the execution of mRNA transport via the sieve elements. The glycine-rich protein 7 (GRP7) was the most abundant RBP in our libraries. Seven KH domain proteins (including Nova), four Pumilio proteins, and all six potato PTBs were identified (Table 5).

Surprisingly, one of the most abundant RBPs was Pumilio1. Pumilio proteins are post-transcriptional regulators containing Puf domains (Pumilo and FBF) that recognize RNA sequences present in the 3′ UTR of target RNAs. Pumilio functions in cytoplasmic de-adenylation, translational repression, RNA localization and decay, maintenance of germline stem cell identity, translation initiation, and rRNA processing and ribosome biogenesis [68]. Puf proteins repress translation of target RNAs during establishment of polarity in the developing embryo of Drosophila and during the localization of Ash1 mRNA to the distal tip of the budding cell [84, 85]. They bind to RNAs at a motif containing a conserved UGUR (where R is a purine). Despite its importance, only a scarcity of information is available on the function of these RBPs in plants [59, 61]. Whereas the Pumilio protein, APUM23, functions in polarity formation in Arabidopsis [61], the role of any Puf proteins in vascular biology is completely unknown.

As previously mentioned, a PTB protein was identified as the core protein in a mobile RNA/protein complex in the phloem of pumpkin [20]. RBP50 has two orthologs in the potato genome, designated StPTB1 and StPTB6. The PTB family of RNA-binding proteins are functional in a wide range of posttranscriptional processes including RNA stability [86], splicing regulation [87], localization [88], translation control [89], and long-distance transport [20]. There are two subfamilies of plant PTBs. One is represented by StPTB1, StPTB6, CmRBP50, and AtPTB3 and these are speculated to be involved in long-distance movement. A second subfamily of PTB proteins, represented by AtPTB1 and −2 and the StPTB7 types, function in alternative splicing [57, 90]. The KH-domain protein, Nova, binds to both StPTB1 and −6 [56] and a Nova ortholog was identified in pumpkin phloem sap [2]. Alba was included because it interacts with the mobile RNA POTH1 [21]. Identification of RBPs and their target RNAs in potato PAC provides a valuable experimental framework for testing interactions between proteins and RNAs that may be functional in long-distance signaling processes. For example, screening for binding elements in RNA sequences comparable to the approach implemented for the PTBs, Nova, and Pumilio in this study could be readily performed for any RBP of interest.

Conclusions

Our results confirm that the combination of laser capture microdissection and RNA-seq provides an invaluable and in-depth approach to the study of phloem biology and a comprehensive picture of the mechanisms associated with long-distance signaling and transport. Out of the roughly 39 k genes in the potato genome, approximately 15 k were expressed in PAC of petiole and stem, numbers that are comparable to the number of genes identified in the vascular bundles of cucumber and watermelon [36]. Our GO analysis indicates that signaling and binding processes are important biological activities associated with phloem cells. The high proportion of RBPs in the expression profiles and the high percentage of transcripts containing binding motifs for three prominent RBPs in their downstream sequences suggest an important role for RNA binding in vascular tissue. The results of this study illustrate the potential of RNA profiling for providing insights into long-distance transport processes mediated by environmental cues that are associated with the sieve element system.

Methods

Plant material

All RNA samples were from the photoperiod-responsive genotype S. tuberosum ssp. andigena. This subspecies only tuberizes under SDs. Plants were propagated in vitro on MS media to root and cultured for 4 weeks before moving to soil. The plants were first transplanted to 7.5-cm square pots and covered with plastic to maintain humidity for a week. After 10–14 days, plants were transferred to 15-cm round pots. The plants were maintained in a growth chamber under a long-day photoperiod (16 h light at 22 °C, 8 h dark at 18 °C, with a fluence rate of 280 μmol m−2 s−1) for 4 weeks before being grown under long-day (16 h light at 22 °C, 8 h dark at 18 °C) or short-day (8 h light at 22 °C, 16 h dark at 18 °C) conditions for 10 more days.

Sample preparation and laser capture microdissection

Two to three-millimeter tissue segments of Solanum tuberosum ssp. andigena plants grown under short-days were excised from the central regions of lower stem internodes (4 to 6 cm above the soil line) or petioles from fully emerged leaves near the top of the plant. At harvest, stem or petiole segments were immersed in at least 10-fold volumes (v/v) of cold fixative (75 % ethanol and 25 % acetic acid) contained in glass vials on ice. Samples were evacuated (0.067 MPa) for 30 min on ice and then fixed 6 h (petioles) or 24 h (stems) in a 4 °C cold room. Tissue segments were transferred to an excess volume of 75 % ethanol (v/v) at 4 °C for 1 h. The process was repeated once to remove excess fixative. Tissues were dehydrated and paraffin-embedded following protocols of Cai and Lashbrook [91]. Multiple tissue segments were arranged vertically in each embedding mold. Metal embedding molds were sequentially washed with xylol and ethanol prior to air-drying and use. Embedded tissues were stored at −20 °C in sealed containers prior to paraffin sectioning. Tissue cross sections (8 μm thick) were cut on a rotary microtome (AO Spencer 820 Microtome; American Optical) using Leica blades. Paraffin sections were stretched for 1 min onto Probe-on Plus slides (Fisher Scientific) containing 5 mM EDTA in DEPC-treated water, pH 8, at 42 °C. Slides were air-dried at RT for up to 5 h before laser microdissection coupled to laser pressure catapulting (LMPC). Immediately before LMPC, slides were deparaffinized twice in xylol for 15 min each and air dried at RT. One-half ml LMPC tubes with clear, non-adhesive caps (Zeiss, Hamburg, Germany) were disinfected prior to use by submerging in chloroform followed by air-drying. For microdissection, the PALM® Laser Microbeam instrument (Bernried, Germany) was employed. A pulsed UV nitrogen laser beam is projected through the objective lens to a narrow diameter (<1.0 μm) that ablates the target without heating adjacent material. Cells were selected using the graphic tools of the PALM RoboSoftware. Laser pressure catapulting (LPC), with a high photonic pressure force, was used to capture the target phloem cells into the lid of a LPC-microfuge tube containing 25 μl extraction buffer from a Picopure RNA kit (Arcturus Engineering, Mountain View, CA, USA). For each sample an area of approximately 1.5 × 106 μm2 comprised of approximately 5000 cells was collected. To minimize degradation, total harvest time was restricted to 1 h per sample. After cell collection, tubes were inverted and the cap end was vortexed in several short spurts to release cells. Contents of the upright tube were pulsed in a microfuge, incubated for 30 min in a water bath at 42 °C, centrifuged at 800 × g for 2 min and stored at −80 °C until RNA isolation.

RNA isolation, library preparation and sequencing

RNA was isolated from microdissected cells with the PicoPure RNA isolation kit (Arcturus Engineering, Mountain View, CA, USA), incorporating an on-column treatment step with RNase-Free DNase (Qiagen, Valencia, CA, USA, Cat #79254). Finally, RNA was quantified with an Agilent 2100 Bioanalyzer using reagents from the manufacturer’s RNA 6000 Pico kit and then stored at −80 °C. Purified RNAs were amplified using Ovation® RNA-Seq system (NuGEN). cDNA libraries were prepared using 2.0 μg of amplified cDNAs and sequenced at the DNA Facility, Iowa State University. For the LD/SD petiole experiment, 2 to 3 cm petiole sections near the junction of the petiole and stem were excised and harvested for both photoperiod conditions. Total RNA was extracted from petioles of long-day or short-day grown S. tuberosum ssp. andigena using RNeasy mini kit (Qiagen). After validation of the quality of RNAs using the 2100 Bioanalyzer, approximately 3.0 μg of total RNA were used for library preparation and sequenced using the HiSeq2500 (Illumina) at the DNA Facility, Iowa State University. The set of LCM isolated samples was sequenced with either Genomic DNA/cDNA/BAC GA II 75-Cycle or mRNA-Seq HiSEQ High Output 100-Cycle. The set of whole petiole samples was sequenced with mRNA-Seq HiSEQ High Output 100-Cycle P.E.

Processing of reads

All the reads were processed as output in fastq format. These reads were aligned to the potato genome (PGSC_DM_v4.03_pseudomolecules.fasta & PGSC_DM_V403_genes.gff) from the potato genome database http://solanaceae.plantbiology.msu.edu/pgsc_download.shtml) with GMAP and GSNAP (http://research-pub.gene.com/gmap/). Parameters were set as default. The number of concordant unique reads in each library was counted with HTseq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html). The disparity of reads that were mapped to the genome in a concordant and unique manner between the LCM-PACs (25 to 47 %, Additional file 1: Table S1) and the whole petiole (~94 %, Additional file 9: Table S8) samples is most likely explained by the fact that the LCM-derived RNA was collected in picogram amounts and subsequently amplified, whereas the whole petiole RNA was not [92]. It would appear that amplification produced reads that map to multiple locations disproportionately over the uniquely mapped reads. Because samples were only compared between the same tissue types (stem PAC vs. pet PAC or LD pet vs. SD pet), no sample bias was introduced.

Statistical analysis

All the libraries were normalized with the 0.75 quantile to eliminate the difference caused by sample scale and sequencing depth. The LCM-derived libraries were sequenced with two different sequencing methods, paired-end and single-end sequencing. Both the difference coming from petiole vs. stem organs and the difference coming from different sequencing methods were considered in the Generalized Linear Model. With the R package “QuasiSeq” [93] (http://cran.rproject.org/web/packages/ QuasiSeq/index.html), quasi-negative binomial deviances of each gene were computed, and the normalized count data was fitted with a quasi-likelihood model. DE genes were selected with adjusted p-values less than 0.2. The p-value was adjusted with the method from Nettleton et al. [94]. Effect from single-end sequencing method is removed based on the derived coefficient. LD- and SD-petiole samples were sequenced with a multiplexing tag in the same lane, so the photoperiod effect is the only effect to be analyzed in comparison. The count of each gene in each sample type is the mean value of the normalized reads of the three or four replicates.

GO analysis and GOSeq

Gene ontology categories of all the genes in potato were obtained from the GO database using Blast2GO (http://www.blast2go.com/b2glaunch/start-blast2go), with parameters of 20 hits and an e-value of 10e−6 (Additional file 20: Table S14). This analysis was performed on the iPlant platform. Gene ontology analysis was performed with GOseq [64] to identify over-represented GO terms in the DE genes. A probability weighting function (PWF) was generated based on transcript length and was applied to eliminate the bias arising from this parameter. The transcript length was obtained from the longest transcript sequence available from the potato genome database (PGSC_DM_v3.4_transcript-update_representative.fasta) for each gene. When the number of over-represented GO terms was too large to visualize, GO terms were reduced with GOslim (http://agbase.msstate.edu/cgi-bin/tools/goslimviewer_select.pl).

Motif search

For the motif search, the potato genome (V4.03) and annotations from the Potato Genome Sequencing Consortium [62, 95] were utilized. The position weight matrices for Nova and Pumilio motifs were obtained from Jiang et al. [67]. We searched the entire potato genome for the presence of these motifs using MEME suite [71]. Because the exact length of the 3′ UTR is currently unavailable, we chose an arbitrary fixed length of 1000 nt for the UTR. For each gene, the 1000 bp region downstream from the end of the coding sequence was considered as its 3′ UTR in this analysis. Finally, using the ‘intersectBed’ tool (BEDTools v2.18.2) [72], we extracted the genes containing Nova or Pumilio motifs in the designated 3′ UTRs. The position of motifs was also identified using the ‘intersectBed’ tool.

Real time RT-PCR

RNA preparation and RT-qPCR were performed as previously described [48]. Primers are listed in Additional file 21: Table S15.

Availability of supporting data

Sequence reads of the LCM RNA-seq and SD/LD petiole RNA-seq have been deposited in NCBI-SRA in a FASTQ file. DOI: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA290800

Abbreviations

CU: 

Cytosine/uracil

DSS: 

Downstream sequence

FD: 

Flowering Locus D

FT: 

Flowering Locus T

GO: 

Gene ontology

LCM: 

Laser capture microdissection

LD: 

Long day

PAC: 

Phloem-associated cells

PTB: 

Polypyrimidine tract-binding protein

RBP: 

RNA-binding protein

SD: 

Short day

UTR: 

Untranslated region

Declarations

Acknowledgements

The authors would like to thank Prof. Jack Horner and Tracey Pepper for their assistance with microscopy work and Mike Baker for his technical help with sequencing. Thanks also to Dr. Peng Jiang for sharing details on their position weight matrixes for the RNA-binding motifs. This research was supported by the NSF Plant Genome Research Program award no. DBI-0820659 and National Research Initiative grant no. 2008–02806 from the USDA National Institute of Food and Agriculture.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Plant Biology, Iowa State University
(2)
Department of Plant Breeding, Genetics, and Biotechnology, Michigan State University
(3)
Office of Biotechnology, Iowa State University

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