Auxin Regulation and MdPIN Expression during Adventitious ... · Auxin and MdPIN S regulate...

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Auxin Regulation and MdPIN Expression during Adventitious Root Initiation in 1

Apple Cuttings 2

Ling Guan1,2, Yingjun Li1, Kaihui Huang1, Zong-Ming (Max) Cheng1,3 3

1 College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China 4 2 Institute of Pomology, Jiangsu Academy of Agricultural Sciences · Jiangsu Key 5 Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014，China 6 3 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37831, USA 7 8 Author for correspondence: 9 10 Zong-Ming (Max) Cheng 11 12 Tel: 86-25-84396055, 1-865-974-7961 13 14 E-mail: [email protected], [email protected] 15 16

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 19, 2020. . https://doi.org/10.1101/2020.03.18.997973doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.18.997973

Auxin and MdPINS regulate adventitious root initiation

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Abstract 20

Adventitious root (AR) formation is critical for the efficient propagation of 21

elite horticultural and forestry crops. Despite decades of research, the cellular 22

processes and molecular mechanisms underlying AR induction in woody plants 23

remains obscure. We examined the details of AR formation in the apple (Malus 24

domestica) M.9 rootstock, the most widely used dwarf rootstock for intensive 25

production, and investigated the role of polar auxin transport in post-embryonic 26

organogenesis. AR formation begins with a series of founder cell divisions and 27

elongation of interfascicular cambium adjacent to vascular tissues. This process 28

was associated with a relatively high indole acetic acid (IAA) content and 29

hydrolysis of starch grains. Exogenous auxin treatment promoted cell division, 30

as well as the proliferation and reorganization of the endoplasmic reticulum and 31

Golgi membrane. By contrast, treatment with the auxin transport inhibitor 32

N-1-naphthylphthalamic acid (NPA) inhibited cell division in the basal region of 33

the cutting and resulted in abnormal cell divisions during early AR formation. In 34

addition, PIN-FORMED (PIN) transcripts were expressed differentially 35

throughout the whole AR development process, with the up-regulation of 36

MdPIN8 and MdPIN10 during induction, an up-regulation of MdPIN4, MdPIN5 37

and MdPIN8 during extension, and an up-regulation of all MdPINs during AR 38

initiation. This research provides a deeper understanding of the cellular and 39

molecular underpinnings of the AR process in woody plants. 40

41

Keywords: Adventitious root; Apple cutting; Auxin; Initiation; MdPIN; Polar auxin 42

transport. 43

44

Introduction 45

Propagation via cuttings is the most economical method for the mass production 46

of horticultural and forestry plants, while simultaneously maintaining desirable 47

genetic traits (Lei et al., 2018a,b). Adventitious root (AR) formation is essential to 48

this process, and its induction has been studied for decades (De Klerk et al., 1995; 49

Dubois et al., 1988). AR formation is affected by many factors including juvenility, 50

ontology, species/genotypes, and various environmental conditions, such as extreme 51

temperatures, salt stress, and/or the content of H2O2, NO, and Ca2+ (Guan et al., 2015), 52


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as well as by the endogenous and exogenous application of plant hormones (Uwe et 53

al., 2016; Guan et al., 2019). Although AR formation has been studied at the 54

anatomical level, its molecular basis, including the mechanism that triggers the 55

process, remains unclear (Bryant et al., 2015). AR formation can be divided into three 56

phases: induction, initiation, and extension, which lead to new visible root systems. 57

AR can initiate from internodes, callus formed at the base of cuttings, or the 58

hypocotyl of herbaceous plants (Li et al., 2009; Lovell and White, 1986). In apple, 59

ARs emerge from lenticels, in which large intracellular spaces allow for gas exchange 60

in stems. 61

According to recent anatomical studies in Arabidopsis thaliana, founder cell 62

division gives rise to AR primordial (ARP), which then develops into a new root 63

(Della Rovere et al., 2013). Similar to that of primary or lateral roots (LRs), the 64

indeterminate growth of AR depends on cell division and elongation, which are 65

affected by various environmental conditions (Nguyen et al., 2018). AR formation is 66

regulated by almost all major plant hormones, with auxin strongly promoting the 67

process (Elmongy et al., 2018). Exogenous auxin treatment or elevated endogenous 68

auxin levels through genetic engineering increase the rate of AR formation and the 69

number of AR formed, whereas impaired auxin signaling or transport via mutagenesis 70

or auxin transport inhibitors inhibit AR initiation (Dai, et al., 2005; Yang et al., 2018; 71

Uwe et al., 2016). 72

Auxin gradient acts as a master controller of AR formation, development, and 73

geotropic responses (Park et al., 2017; Guan et al., 2019). The relationship between 74

the post-embryonic root of AR (and LR) formation and polar auxin transport has been 75

established by using the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) 76

(Agulló-Antón et al., 2014; Liu et al., 2009). The promoting effect of polar auxin 77

transport (PAT) on AR development can be counteracted by NPA, which disturbs 78

polar auxin transport and consequently, AR formation (Mignolli et al., 2017). 79

The asymmetric cellular localization of PIN-FORMED (PIN) auxin efflux 80

carriers have been shown to play a rate-limiting role (Weller et al., 2017) in directing 81

cell-to-cell auxin flow (Forestan and Varotto, 2012; Zažímalová et al., 2007). Despite 82

their importance, the roles of auxin and PINs in regulating this process in woody 83

horticultural plants have rarely been studied. Here, we integrated anatomy and 84

ultrastructural observation, hormone analysis, and the spatiotemporal expression 85


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profile of the PIN genes to investigate the details of early initiation and organization 86

of AR primordia within the lenticels of (Malus domestica) rootstock M.9 cuttings. 87

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89

Materials and methods 90

Plant material and growth conditions 91

Six-month-old apple M.9 cuttings were used for all studies. The bases of the 92

cuttings (0-4 cm) were submerged in ventilating hydroponic containers filled with 93

Hoagland’s nutrient solution, pH 5.8 (Eliasson, 1978), and allowed to grow in a 94

growth chamber (~25 °C) under a 16:8 L/D cycle with 300 μmol·m-2·s-1. All plant 95

samples were collected at 10:00 AM each day and were harvested every 24 hours for 96

168 hours (7 days). Plant materials used for morphological studies were fixed in 2.5% 97

glutaraldehyde, and those used for phytohormone quantitation and gene expression 98

analysis were frozen in liquid nitrogen and stored at -80 °C until use. 99

100

Treatments and growth analysis 101

Plants were grown in Hoagland’s nutrient solution (control) or supplemented 102

with 10 μM indole-3-acetic acid (IAA) or N-1-naphthylphthalamic acid (NPA), as 103

positive and negative regulators of polar auxin transport, respectively. IAA and NPA 104

were purchased from Sigma-Aldrich (catalog numbers 12886 and 33371, 105

respectively), and were dissolved in dimethyl sulfoxide. 106

107

Anatomical and ultrastructural observation 108

Cryosectioning was used to observe anatomical changes during AR initiation. 109

Samples were dehydrated and fixed in 2.5% glutaraldehyde solution according to 110

Chen et al. (1985). The division and elongation of AR founder cells were observed 111

with a stereoscopic microscope (Leica MZ 6; Wetzlar, Germany) and ImageJ (v. IJ 112

1.46r; Schneider et al. 2012) was used for data capture and analysis (Ferreira, 2012). 113

Statistical differences in AR density and percent elongation were determined by 114

ANOVA with Tukey’s post hoc test in SPSS 10 (Meulman, 2000) with p <0.05. 115

Cryosectional samples were also used for scanning electron microscopy (SEM) and 116

transmission electron (TEM) microscopy. 117

118


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Quantitation of free phytohormones 119

Submerged apple cuttings with approximately equal weight, length, and diameter 120

for each treatment were collected, and 5-mm segments from the bases of the cuttings 121

were used to determine IAA and zeatin (ZT) contents. The samples were incubated in 122

an ice-cold uptake buffer (1.5% sucrose, 23 mM MES, pH 5.5) for 15 min, followed 123

by two 15-min washes with fresh uptake buffer. The cleared tissue was surface-dried 124

on filter paper and then weighed. IAA and ZT levels were measured using 125

HPLC–ESI–MS/MS; Pan et al., 2010). The reverse-phase HPLC gradient parameters 126

and selected reaction monitoring conditions for protonated or deprotonated plant 127

hormones ([M + H] + or [M − H] −) are listed in supplementary Tables S1 and S2. 128

IAA and ZT standards were purchased from Sigma (catalog numbers 12886 and 129

Z0750, respectively). 130

131

RNA extraction and qPCR 132

RNA was extracted using CTAB, and 1 μL was used as the template for cDNA 133

synthesis using the TaKaRa PrimerScript RT Reagent Kit (RR037B; Liaoning, China). 134

Quantitative PCR (qPCR) was performed with an ABI PRISM 7900HT system 135

(Applied Biosystems; Foster City, CA, USA) using a 10-fold dilution of the cDNA 136

template. The reaction mixture included 5 μL of template, 7.5 μL of SYBR Green 137

PCR master mix (4309155; Applied Biosystems), and l μM of each of the two 138

MdPIN-specific primers (Tables S3, S4) in a final volume of 15 μL. Primer efficiency 139

was determined with a standard curve analysis using 5-fold serial dilution of a known 140

amount of template, and amplicon specificity was confirmed by sequencing. The 141

thermal cycle regime consisted of 2 min at 50 °C, 10 min at 95 °C, followed by 40 142

cycles of 15 sec at 95 °C, 30 sec at 54 °C, and 30 sec at 72 °C. Disassociation curves 143

and gel electrophoresis were used to verify the amplification of a single product. CT 144

values were calculated using the SDS2.1 software (Applied Biosystems) and data 145

were analyzed using the 2ΔΔCT method with 18S rDNA as a reference gene for 146

normalization (Livak and Schmittgen, 2001). 147

148

Results 149

AR initiation in apple cuttings 150


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In order to determine the exact position of AR formation, six-month-old M.9 151

apple cuttings were cultured in Hoagland’s nutrient solution. The submerged stems 152

were analyzed for AR formation over a seven-day period (168h), during which the 153

lenticels continuously expanded (Fig. 1A, C and F). Noticeable lenticel enlargement 154

was seen at 72h, and new adventitious roots emerging from the lenticels were easily 155

observed as small protrusions at 168h (Fig. 1F). The SEM analysis revealed 156

progressive longitudinal splitting of the submerged lenticels in the controls until a 157

deep fissure was observed at 168h (Fig. 1H, J, M, O, Q and T). IAA treatment 158

accelerated longitudinal splitting in lenticels at each time point. For example, 159

compared with the corresponding control, the lenticel dehiscence became deeper at 160

72h (Fig. 1K, R) and the lenticel surface was almost completely disrupted at 168h 161

(Fig. 1N, U). By contrast, lenticel longitudinal splitting was very thin and shallow in 162

NPA-treated cuttings at 72h (Fig. 1I, P), and the fissures were still very narrow and 163

shallow at 168h as compared to control cuttings (Fig. 1L, S). 164

The cells underlying the longitudinal splitting in the lenticels were examined to 165

better understand the dehiscence process. The cambial zone in control cuttings had 166

6–8 layers of cells between the xylem and phloem, and a large number of 167

parenchymatous cells were observed in the interfascicular cambium next to the 168

vascular cylinder (Fig. 2A, E; Fig. S1). During the 72-168h time interval, founder cell 169

divisions produced a large number of cells in the interfascicular cambium adjacent to 170

vascular tissues (Fig. 2A, E, C, G, J, M, P and S). These founder cells exhibited dense 171

cytoplasm and swollen nuclei (Fig. S2), features that are indicative of meristematic 172

activity. Histological observations revealed differences in the founder cells underlying 173

the lenticels in NPA- and IAA-treated cuttings compared with controls. Specifically, 174

NPA-treated lenticels displayed fewer dividing founder cells at 72h and 168h (Fig. 2B, 175

F, I, L, O and R) compared with corresponding controls. By contrast, the lenticels of 176

IAA-treated apple cuttings had more founder and parenchymatous cells at 72 h (Fig. 177

2B-D, F-H) and more founder cells in the interfascicular cambium at 120h compared 178

with the controls (Fig. 3A), and this process continued through 168h (Fig. 2O-P, R-S). 179

At 168h, the number of elongated founder cells under lenticels continued to increase 180

and started to protrude from the lenticel surface in IAA-treated apple cuttings (Fig. 181

2Q, T). Together, these results indicate that lenticel dehiscence and AR protrusion 182


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begin by 72h and 168h, respectively, and that IAA promotes lenticel longitudinal 183

splitting during early AR emergence. 184

185

Auxin controls founder cell division and elongation 186

By 168h, the density ratio of divided founder cells to total parenchymal cells was 187

two-fold greater in IAA-treated apple cuttings over the control (Fig. 3A). This time 188

point also exhibited the highest number of divided founder cells in the IAA-treated 189

cuttings (Fig. 3A). Taken together, these data suggest that IAA stimulates founder cell 190

division. By contrast, the density of divided founder cells in NPA-treated apple 191

cuttings was approximately half of that of the control cuttings between 72h to 144h 192

(Fig. 3A); further, at 168h, the density of divided founder cells in NPA-treated apple 193

cuttings was 0.75, comparable to that in the control at 144h (Fig. 3A). These 194

observations suggest that NPA delayed founder cell division during late AR initiation. 195

Overall, the density of divided founder cells increased over time in all conditions 196

tested, with IAA and NPA treatments stimulating and inhibiting founder cell division, 197

respectively. 198

Elongated founder cells were first observed at 72h in IAA-treated cuttings, 24 199

hours prior to their appearance in the controls (Fig. 3B). Interestingly, elongated 200

founder cells were also observed at 96h in NPA-treated cuttings, but in a lower 201

proportion as compared to the control (Fig. 3B). Like those above, these data support 202

the idea that IAA and NPA stimulate and inhibit founder cell division, respectively. 203

At 168h, elongated founder cells accounted for 16.9%, 35.5%, and 8.8% of total cells 204

in the control, IAA-, and NPA-treated cuttings, respectively. These data are consistent 205

with the changes in divided founder cell density in the respective treatments from 96 206

to 168h (Fig. 3A), suggesting that AR formation in apple is developmentally 207

programmed. 208

209

Organelle and endomembrane changes during AR initiation 210

To further investigate the morphological and physiological changes during AR 211

initiation, subcellular structures were examined with transmission electron 212

microscopy (TEM). Numerous starch grains were observed in the plastids at the 0h 213

and 24h time points for all treatments (Fig. 4A-I). However, at 72h, the number and 214

density of starch grains rapidly decreased from the plastids of control and IAA-treated 215


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cells, but little change in the density was observed in the plastids of NPA-treated cells 216

(Fig. 4J-L, P-R). At 72h, no changes in starch grain content or organelles were 217

observed in unsubmerged stems (Fig. S3). 218

The number and density of the mitochondria, endoplasmic reticulum, and Golgi 219

apparatus in IAA-treated cells were higher than those in control at 72h (Fig. S4). The 220

number of elongated founder cells in control and IAA-treated cuttings continued to 221

increase from 72 (Fig. 4Q-R) to 168h (Fig. 4M-O, S-U). By contrast, many dead cells, 222

and cells with abnormal nuclei, were observed in NPA-treated cuttings (Fig. S4). 223

These observations, taken together with the near constant level of starch grains noted 224

in NPA-treated cuttings, the plausible explanation emerges that NPA inhibits starch 225

grain degradation, thus blocking energy needed for founder cell division and 226

elongation, resulting in abnormal subcellular structures and cell death. 227

228

Auxin and cytokinin contents change in a complementary manner 229

Auxin and cytokinin are both known to play key roles in AR initiation and 230

elongation (Guan et al., 2015). Therefore, we determined the changes in the 231

concentrations of IAA and zeatin (ZT) during AR initiation in control, and IAA- and 232

NPA-treated cuttings (Fig. 5). Although the IAA concentrations differed between 233

treatments, the trends in the changes of IAA levels were consistent between 234

treatments during the test period. For example, IAA concentration increased steadily 235

with time from 48h to120h in all three treatments and peaked at 120h with 1156.4 236

ng/g FW, 1216.6 ng/g FW, and 572.89 ng/g FW, in control, IAA- and NPA-treatment, 237

respectively (Fig. 5A). At 168h, IAA level in control and NPA-treated cuttings 238

reduced significantly to 707.2 ng/g FW and 338.8 ng/g FW, respectively, but reduced 239

only slightly to 1137.6 ng/g FW in IAA-treated cuttings, compared with those at 240

120h. 241

In control and NPA-treated cuttings, ZT showed a three-phase accumulation 242

pattern with increasing, then decreasing, and finally increasing again by the end of the 243

experimental period (Fig. 5B). In the control and NPA-treated cuttings, ZT levels 244

increased from 48-96h, decreased at 120h, and increased again at 168h (Fig. 5B). In 245

contrast, this drop was not observed in IAA-treated cuttings, but instead leveled out 246

between time points 96 and 120h, with a significant increase in ZT levels at 120h. 247

Overall, NPA-treated cuttings exhibited lower ZT levels compared to that of the 248


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control. For example, ZT levels in NPA-treated cuttings were 36.84% and 72.50% 249

lower than those in control cuttings at 120 and 168h, respectively (Fig. 5B). However, 250

the control and NPA-treated cuttings shared a similar ZT accumulation pattern, which 251

well correlated with that of the change in IAA levels (Fig. 5A). In contrast, ZT levels 252

in IAA-treated cuttings increased with time with no drop in concentration and peaked 253

at 1080.7 ng/g FW at 168h (Fig. 5B), two-fold of that in the control cuttings at 168h. 254

255

MdPIN expression is stage-specific 256

To correlate MdPIN gene expression with the morphological and hormonal 257

changes associated with AR in the apple cuttings, we determined the transcription 258

profiles of the eight MdPIN family members at each major stage of AR formation. All 259

eight MdPIN gene members were expressed during all three phases of AR formation 260

but exhibited distinct spatiotemporal expression patterns (Fig. 6). For example, 261

MdPIN1 expression in control cuttings increased from 0h through 96h and peaked at a 262

level 7.2-fold of that at 0h. At 96h, MdPIN1 expression in control and IAA-treated 263

cuttings was 1.4-fold and 11.2-fold of that at 0h, respectively. In NPA- and 264

IAA-treated cuttings, MdPIN1 expression showed the same decreasing trend at 265

subsequent time points (Fig. 6A). 266

MdPIN2 expression showed relatively uniform expression patterns across the 267

time course, and the fold changes of MdPIN2 expression between 24h-168h compared 268

with that at 0h ranged from 1.5-2-fold in control and NPA-treated cuttings. MdPIN2 269

expression was IAA responsive and increased by up to 5-fold through 24h to 168h 270

(Fig. 6 B). NPA treatment decreased MdPIN2 expression at all time points. 271

MdPIN3 was expressed at relatively low levels during the time course (Fig. 6 C). 272

The expression levels peaked at 96h in control and IAA-treated cuttings and were 273

about 1.8- and 4.3-fold of that at 0h, respectively. In addition, MdPIN3 expression 274

was significantly inhibited by NPA at all time points, whereas IAA treatment 275

stimulated MdPIN3 expression, especially from 144h through 168h (Fig. 6 C). 276

MdPIN4 expression peaked at 96h and 168h, with an approximately 4-fold 277

increase compared with that at 0h (Fig. 6D). MdPIN4 expression appeared to be 278

sensitive to IAA and NPA treatments after 48h, with the highest expression levels 279

appearing at 96h 2.2- and 10.2-fold higher than that at 0h in NPA- and IAA-treated 280

cuttings, respectively. 281


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MdPIN5 had the highest level of expression in the control treatment at 72h and 282

96h, with a 13.5-fold increase at 96h in comparison of that at 0h (Fig. 6E). MdPIN5 283

expression was IAA sensitive and its expression increased in the same pattern 284

observed for controls, with a 18.2-fold increase at 96h. NPA treatment strongly 285

inhibited MdPIN5 expression in all time points. 286

MdPIN7 expression peaked at 24h and 120h, approximately 2.5-3-fold of that at 287

0h (Fig. 6F). MdPIN7 expression was sensitive to IAA starting at 72h and expression 288

increased to 5.3-fold at 168h. MdPIN7 showed little response to NPA treatment. 289

MdPIN8 expression increased 3-fold at 24h compared to the baseline at 0h, and 290

IAA treatment induced MdPIN8 expression during the time course (Fig. 6 G). In 291

contrast, NPA slightly inhibited MdPIN8 expression. Relatively high expression of 292

MdPIN8 appeared at 72h and 168h, corresponding to the initiation phase. MdPIN8 293

expression peaked at 168h, with 5.5 times of that at 0h. In NPA- and IAA-treated 294

cuttings, MdPIN8 expression peaked at 72h, with 4.9- and 7.2-fold of that at 0h, 295

respectively. From 96h-168h, NPA treatment increased MdPIN8 expression. 296

MdPIN10 was also expressed during the early stages of AR formation, peaking 297

at 48h, 2-fold above the baseline. At 48h, MdPIN10 expression in NPA- and 298

IAA-treated cuttings increased by 3.4- and 5.6-fold, respectively, compared with the 299

levels at 0h, (Fig. 6H). In IAA-treated cuttings, MdPIN10 expression peaked at 300

8.6-fold at 96h. In contrast to MdPIN8, MdPIN10 expression was inhibited by NPA at 301

all time points. 302

303

Discussion 304

AR formation requires the initiation of new founder cells from interfascicular 305

cambium adjacent to vascular tissues 306

AR formation is crucial for commercial propagation, and depending on the 307

species, there are two mechanisms for AR formation in most woody plants. AR 308

founder cells can either 1) initiate in the stem but remain dormant until the induction 309

of AR formation by environmental conditions (Guan et al., 2015), or 2) initiate de 310

novo from cells, such as phloem or xylem parenchyma cells, within or adjacent to the 311

vascular tissues, such as cells from interfascicular cambium or the phloem/cambium 312

junction (De Klerk et al., 1995; Naija et al., 2008; Guan et al., 2015). A typical apple 313

stem contains collateral vascular bundles with a ring around the pith (Figs. 1 and 2, 314


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Fig. S1). Initially (at 0h), the cells underlying the lenticels in the interfascicular 315

cambium adjacent to vascular tissues had no observable meristematic activity, 316

whereas the founder cells had already undergone numerous cell divisions 72h-168h 317

post-cutting (Fig. 2, Fig. S2). Therefore, AR formation in apple rootstock M.9 318

cuttings occurs via the second mechanism. This is consistent with that reported 319

recently in forest tree species, especially conifers, that roots are induced from 320

determined or differentiated cells, and further from positions where roots do not 321

normally occur during development (Pizarro et al., 2018). 322

323

ARs protrude through lenticels 324

Lenticel formation culminates with the disintegration of cork cells under the cork 325

cambium that are ultimately replaced by loosely arranged thin-walled cells, also 326

called supplemental cells or filling cells, during early cork layer formation. Later on, 327

epidermis and cork are squeezed until they crack as supplemental cells continue to 328

divide, and eventually split into a series of lip projections, the lenticels (Topa and 329

McLeod, 1986). The tissues inside lenticels provide a natural channel, for not only 330

gas exchange during flooding or other abiotic stress, but also for AR formation and 331

growth. In this study, lenticels of apple cuttings exhibited an increasingly larger crack 332

during early AR formation (Fig.1A-G). Our results showed that at each time point 333

during AR development, the lenticels of control and IAA-treated cuttings were more 334

severely ruptured by founder cell division compared with those of the NPA-treated 335

cuttings (Fig.1H-U). These developmental changes indicate that auxin promotes 336

lenticel dehiscence and AR protrusion from lenticel channels. This process is similar 337

to auxin-mediated channel formation for lateral root (LR) emergence in Arabidopsis 338

thaliana (Swarup et al., 2008; Stoeckle et al., 2018). In both cases, auxin uptake into 339

cortical and epidermal cells overlying the LR primordium during emergence results in 340

increased expression of genes encoding cell wall-remodeling enzymes, such as 341

pectate lyase, to promote cell separation for LR emergence (Swarup et al., 2008; 342

Stoeckle et al., 2018). In addition, histological observations detected intra-stem 343

channels formed by enlarged cavities in the interfascicular cambium, and the number 344

and dimensions of these cavities increased over time (Fig. 2). These results further 345

suggest that the cells located in front of newly formed founder cells might have 346

undergone programmed cell death (PCD) to allow AR primordium development, 347


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supporting the conclusion of a previous study on AR formation in tomato (Steffens 348

and Sauter, 2005). 349

350

The induction phase of AR formation in apple cuttings 351

AR formation in apple cuttings can be divided into three phases based on 352

characteristic cellular changes as we described in the anatomical observations: 353

induction (0-72h), initiation (72-120h), and extension (120-168h). In control and 354

IAA-treated cuttings, the induction phase at 72h was characterized by an increased 355

number of founder cells with dense cytoplasm and swallow nuclei (Fig.2 G-H), as 356

well as the hydrolysis of starch grains (Fig. 3 P-Q), which presumably provided 357

energy for the further division and elongation of AR founder cells. By contrast, a 358

large number of starch grains remained in the chloroplasts at 72h in NPA-treated 359

apple cuttings (Fig.4 J), suggesting that NPA had inhibited starch hydrolysis during 360

AR formation whereas IAA enhanced the conversion of starch into “cash energy” for 361

AR induction. These findings suggest that IAA modulates AR induction by inducing 362

the dedifferentiation of interfascicular cambium cells into AR founder cells. 363

Therefore, AR initiation begins with the division and elongation of a large 364

number of clustered founder cells that fill the cavity opened by lenticel splitting in 365

control and IAA-treated cuttings (Fig.2M-N; Fig. 3F-N), whereas founder cell 366

division was severely impaired in NPA-treated apple cuttings (Fig.2 L). In addition, 367

IAA treatment significantly increased the ratio of divided founder cells to total 368

parenchymal cells, and founder cell density increased more quickly than in controls 369

(Fig. 3A). These data demonstrate the promoting effect of IAA on primordial founder 370

cell division upon the induction of AR formation (Fig. 3A). 371

With elongation of the founder cells, AR formation advances into the extension 372

phase when the percentage of elongated cells among divided founder cells increased 373

(Fig. 4P-Q, S-T). Elongated AR founder cells appeared earlier in IAA-treated cuttings 374

and were almost twice as abundant as those in control cuttings (Fig. 3B). Thus, we 375

speculate that both lenticel dehiscence and intra-stem PCD (Steffens and Sauter, 2005) 376

might regulate AR formation by modulating polar auxin transport, which regulates 377

auxin gradient. 378

379

Starch grain depletion is associated with AR initiation 380


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Previous studies have found that maintaining an appropriate auxin level and gradient 381

in the basal portion of shoots is essential for AR formation (Kitomi et al., 2008, 382

Agulló-Antón et al., 2014; Xu et al., 2017) but the exact mechanism underlying this is 383

still unclear. Here, we observed rapid starch grain depletion upon the initiation of AR 384

formation in apple cuttings. It has been proposed that starch accumulation and 385

depletion may be a biochemical indicator for early root formation in hypocotyl 386

cuttings, which provides energy for AR formation (references). This energy 387

supplementation process is positively regulated by auxin (Jásik et al., 1997; Li et al., 388

2000; Husen et al., 2007; Husen et al., 2017). In agreement with the published data, 389

our temporal and spatial analysis study also supports a key role of starch grain 390

accumulation and degradation in AR of woody plants, with IAA exerting a 391

stimulatory effect on AR formation and NPA delaying the process (Fig. 4). In 392

addition, starch grain depletion was also associated with endomembrane proliferation 393

and reorganization (Fig. 4 and Fig. S4). Although the physiological basis of how the 394

two processes relate to AR formation, the fact that grain depletion and new membrane 395

system formation were promoted by IAA and inhibited by NPA suggests that polar 396

auxin transport is the initial driving force for these processes (Yu et al., 2016). 397

398

Roles of auxin and cytokinin during AR formation 399

Recent studies have shown that ZT can suppress adventitious root primordium 400

formation whereas auxin signaling positively regulates this complex biological 401

process, suggesting that relatively high IAA and low ZT levels are prerequisites for 402

AR induction and initiation (Li et al., 2018; Mao et al., 2018). Cytokinins and auxin 403

seem to exert opposite effects on AR formation (Dubois et al., 1988; Yang et al., 404

2017). In addition, auxin can directly down-regulate cytokinin biosynthesis while 405

cytokinins have little effect on auxin biosynthesis (Nordström et al., 2004). The initial 406

high levels of auxin in cuttings at the induction and initiation phases of AR formation 407

were replaced by high levels of cytokinin in the extension phase, suggesting a 408

crosstalk between auxin and cytokinin metabolism, although the molecular basis 409

remains to be characterized. 410

Auxin appears to regulate AR formation mainly by modulating AR founder cell 411

division and elongation during the induction and initiation stages (Fig. 2, 3). Similar 412

to that had been observed for LR formation in Arabidopsis thaliana (Petersson et al., 413


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14

2009), IAA levels in apple cuttings continued to increase during the induction (0-72h) 414

and initiation (72-120h) phases, maximizing at 120h, followed by a steady reduction 415

after entering the elongation phase (120-168h). ZT levels also increased during the 416

induction and initiation phases and peaked at 96h before they started to decrease. By 417

contrast, NPA hampered IAA accumulation but had little effect on ZT levels. These 418

results point to the maintenance of auxin and cytokinin homeostasis via feedback 419

regulation. 420

421

Correlations between MdPIN expression and AR formation 422

Most PINs function in directional auxin transport while displaying differential 423

expression patterns, reflecting their potential multifaceted roles in plant development 424

(He et al., 2017; Short et al.; 2018; Wang et al., 2017). In Arabidopsis, members of 425

the PIN gene family are shown to participate in vascular polar auxin transport, root 426

patterning, root gravitropism, sink-driven auxin gradient establishment, and 427

apical-basal polarity (Friml et al., 2002a; Friml et al., 2002b; Friml et al., 2003; 428

Gälweiler et al., 1998; Mravec et al., 2009; Muller et al., 1998). The PIN family in 429

apple has eight members, all of which are expressed differntially during AR formation 430

(Fig. 6). Therefore, we proposed a possible model of the regulatory mechanism to 431

map the main function of each MdPIN during the rooting of apple cuttings (Fig. 7A). 432

Up-regulation of MdPIN8 and MdPIN10 was mainly associated in AR induction. 433

The initiation phase was associated with an up-regulation of MdPIN1, MdPIN4, 434

MdPIN5, MdPIN8, and MdPIN10; MdPIN3 is up-regulated at the early stage of 435

initiation, and MdPIN2 and MdPIN7 were up-regulated toward the very end of the 436

initiation stage (Fig. 7A). The extension phase is mainly associated with an 437

up-regulation of MdPIN4, MdPIN5, and MdPIN8 (Fig. 7A). Our data suggest that the 438

eight MdPINs likely play different roles throughout the AR process, where their gene 439

products may directly or indirectly regulate AR formation in a cooperative manner by 440

mediating anatomical and physiological changes in the cuttings (Fig. 7B). Future 441

biochemical studies should focus on the diverse expression patterns of MdPIN 442

proteins to further investigate the function of each in regulating this post-embryonic 443

rooting process. 444

445

Conclusions 446


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15

AR formation in apple is a coordinated developmental process. IAA stimulates 447

the process likely via the up-regulation of PIN gene expression. Auxin stimulates AR 448

founder cells division and elongation probably by promoting energy supplementation 449

via starch grain hydrolysis, which leads to endomembrane system proliferation, 450

lenticel dehiscence, and AR emergence in the apple M.9 rootstock. 451

452

Acknowledgements 453

We sincerely thank Yi Li from University of Connecticut and Dr. Angus Murphy, 454

Wendy Peer and Jun Zhang from University of Maryland for their valuable 455

suggestions. We thank Jun Hu, Jin Wang, and Zuli Gu for helping with the 456

experimental material and data collection in this study. This work was partially 457

supported by the National Natural Science Foundation of China (Grant no. 458

31601738). 459

460

Conflict of interest 461

All authors declare no competing interest. 462

Figure legends 463

Fig. 1, Morphological changes in lenticels upon NPA and IAA treatments. 464

Apple cuttings were cultured in Hoagland’s solution and sampled every 24 hours (h) 465

from 0-168h. Three distinctive development phases were observed at 0, 72, and 168h. 466

A-G: physical appearance of submerged cuttings. Arrows point to the origination of 467

new ARs. H-U, SEM micrographs of lenticels in different AR developmental phases; 468

Bar, A-G=0.4cm, H-U=100 μm, the bar I=J=K; L=M=N; P=Q=R; S=T=U. 469

Fig. 2, Anatomical observations of lenticels during the rooting of apple cuttings. 470

Each lenticel was observed under the stereomicroscope at 0h (A, E), 72h (B-D, F-H), 471

120h (I-N), and 168h (O-T). Treatments are: control (A, E, C, G, J, M, P, S), NPA (B, 472

F, I, L, O, R), IAA (D, H, K, N, Q, T). Scale bars, 50 μm. Inset scale bars, 473

A-D=O-Q=0.5 mm. Arrows: yellow, proliferated founder cells; pink, epidermis; 474


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16

brown, parenchyma cells located in interfascicular cambium; dark blue, vascular 475

tissues; red, cavities. 476

Fig. 3, The density and elongation of founder cells during different AR 477

developmental stages. 478

(A). The density of divided founder cells in control, NPA- and IAA-treated apple 479

cuttings. Data are the ratio of divided founder cells to the total number of 480

parenchymal cells. (B). Percentage of elongated cells in control, and NPA- and 481

IAA-treated cuttings. Data are the percentage of the total elongated cells divided by 482

founder cells that have undergone cell division. Elongated cells were at least two 483

times longer than the shorter axis. Error bar represents +/- standard error (SE), n≧15. 484

Bars with different letters indicate statistical significance (p < 0.05). 485

Fig. 4, Ultrastructural changes during the rooting of apple cuttings. 486

Transmission electron microscopy of founder cells at 0h (A-C), 24h (D-I), 72h (J-L, 487

P-R), and 168h (M-O, S-U) during AR development. Shown are control (A-C, E, H, 488

K, Q, N, T), NPA treatment (D, G, J, P, M, S), and IAA treatment (F, I, L, R, O, U). 489

Scale bars denote 0.5 μm in A-C, 1.0 μm in N, D, F, G-J, M, P, S and T, 2.5 μm in E 490

and L; 0.25 μm in K and O; 5 μm in Q, R, and U. Orange arrows, starch grains (S); 491

blue arrows, endoplasmic reticulum (ER); green arrows, elongated cells (EC); red 492

arrows, mitochondria (M). 493

Fig. 5, Indole acetic acid (IAA) and zeatin (ZT) levels during different 494

developmental stages of rooting in apple cuttings. 495

Error bar represents +/- SE, n≧3. Different letters indicate significant differences (p < 496

0.05). 497

Fig. 6, MdPIN gene expression. 498

The gene expression levels of MdPINs were quantified by qPCR in the bases of apple 499

cuttings during different stages of adventitious root formation. The average relative 500

transcript level of each gene was calculated using the 2−ΔΔCT method; data are shown 501

as mean ± SE (n=3). 502


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17

Fig. 7, Stage-specific MdPIN gene expression patterns and a model of cellular 503

regulatory mechanism during AR formation. 504

(A) Differential expression of MdPINs during AR induction, initiation and extension. 505

Dashed lines represent 24h time points. MdPIN8 expression peaked during the 506

induction phase (24h), indicating that MdPIN8 may play a major role in the early 507

stage of AR induction. MdPIN10 expression peaked at 48h, and was mostly low at 508

other time points, suggesting that MdPIN10 also contributes to the induction and 509

initiation of AR. The maximum expression of MdPIN1, MdPIN3, MdPIN4, MdPIN5 510

occurred at 96h, just 24h prior to the morphological changes in the initiation phase, 511

which ends at 120 h. Further, MdPIN1, MdPIN2, MdPIN4, and MdPIN5 expression 512

remained high at 120h, indicating that all of these genes also function during the late 513

stage of initiation. MdPIN7 expression peaked in 120h, suggesting that MdPIN7 may 514

mainly function in the late stage of initiation. At 168h, differential increases in 515

MdPIN4, MdPIN5, and MdPIN8 expression were observed, suggesting that these 516

members promote lenticel dehiscence and AR protrusion from the epidermis in the 517

extension phase. 518

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Fig. 1, Morphological changes in lenticels upon NPA and IAA treatments.

Apple cuttings were cultured in Hoagland’s solution and sampled every 24 hours (h) from 0-168h.

Three distinctive development phases were observed at 0, 72, and 168h. A-G: physical appearance

of submerged cuttings. Arrows point to the origination of new ARs. H-U, SEM micrographs of

lenticels in different AR developmental phases; Bar, A-G=0.4cm, H-U=100 μm, the bar I=J=K;

L=M=N; P=Q=R; S=T=U.

Control Control ControlNPA NPAIAA IAA

0h 72h 168h


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Fig. 2, Anatomical observations of lenticels during the rooting of apple cuttings.

Each lenticel was observed under the stereomicroscope at 0h (A, E), 72h (B-D, F-H), 120h (I-N),

and 168h (O-T). Treatments are: control (A, E, C, G, J, M, P, S), NPA (B, F, I, L, O, R), IAA (D, H,

K, N, Q, T). Scale bars, 50 μm. Inset scale bars, A-D=O-Q=0.5 mm. Arrows: yellow, proliferated


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founder cells; pink, epidermis; brown, parenchyma cells located in interfascicular cambium; dark

blue, vascular tissues; red, cavities.


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Fig. 3, The density and elongation of founder cells during different AR developmental stages.

(A). The density of divided founder cells in control, NPA- and IAA-treated apple cuttings. Data

are the ratio of divided founder cells to the total number of parenchymal cells. (B). Percentage of

elongated cells in control, and NPA- and IAA-treated cuttings. Data are the percentage of the total

elongated cells divided by founder cells that have undergone cell division. Elongated cells were at

least two times longer than the shorter axis. Error bar represents +/- standard error (SE), n≧15.

Bars with different letters indicate statistical significance (p < 0.05).

A B


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Fig. 4, Ultrastructural changes during the rooting of apple cuttings.

Transmission electron microscopy of founder cells at 0h (A-C), 24h (D-I), 72h (J-L, P-R), and

168h (M-O, S-U) during AR development. Shown are control (A-C, E, H, K, Q, N, T), NPA

treatment (D, G, J, P, M, S), and IAA treatment (F, I, L, R, O, U). Scale bars denote 0.5 μm in A-C,

1.0 μm in N, D, F, G-J, M, P, S and T, 2.5 μm in E and L; 0.25 μm in K and O; 5 μm in Q, R, and

U. Orange arrows, starch grains (S); blue arrows, endoplasmic reticulum (ER); green arrows,

elongated cells (EC); red arrows, mitochondria (M).

24h

72h

168h

S

S S S

S

S

S

S

S

S S

S

S

S ER

ER

S

EC EC M

M

EC


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Fig. 5, Indole acetic acid (IAA) and zeatin (ZT) levels during different developmental stages

of rooting in apple cuttings.

Error bar represents +/- SE, n≧3. Different letters indicate significant differences (p < 0.05).

b

c

b

b

c

a

b

a

a

b

a

a

a

aa

0

250

500

750

1000

1250

48 72 96 120 168

IAA content (ng·g-1 FW)

Time (hours)

NPA

Control

IAA

b

b b

bc

b

b

b

b

b

a

aa

a

a

0

200

400

600

800

1000

1200

48 72 96 120 168

ZT content (ng·g-1 FW)

Time (hours)

NPA

Control

IAA

A B


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Fig. 6, MdPIN gene expression.

The gene expression levels of MdPINs were quantified by qPCR in the bases of apple cuttings

during different stages of adventitious root formation. The average relative transcript level of each

gene was calculated using the 2−ΔΔCT method; data are shown as mean ± SE (n=3).


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Fig. 7, Stage-specific MdPIN gene expression patterns and a model of cellular regulatory

mechanism during AR formation.

(A) Differential expression of MdPINs during AR induction, initiation and extension. Dashed lines

represent 24h time points. MdPIN8 expression peaked during the induction phase (24h), indicating

that MdPIN8 may play a major role in the early stage of AR induction. MdPIN10 expression

peaked at 48h, and was mostly low at other time points, suggesting that MdPIN10 also contributes

to the induction and initiation of AR. The maximum expression of MdPIN1, MdPIN3, MdPIN4,

MdPIN5 occurred at 96h, just 24h prior to the morphological changes in the initiation phase,

which ends at 120 h. Further, MdPIN1, MdPIN2, MdPIN4, and MdPIN5 expression remained high

at 120h, indicating that all of these genes also function during the late stage of initiation. MdPIN7

expression peaked in 120h, suggesting that MdPIN7 may mainly function in the late stage of

initiation. At 168h, differential increases in MdPIN4, MdPIN5, and MdPIN8 expression were

observed, suggesting that these members promote lenticel dehiscence and AR protrusion from the

epidermis in the extension phase.

A

B


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(B) A possible model of cellular regulatory mechanisms during AR formation stages.

ER-endoplasmic reticulum; PAT-polar auxin transport; PCD, programmed cell death. Arrows

represent the positive regulatory elements. A line ending with a bar represents negative regulatory

elements.


https://doi.org/10.1101/2020.03.18.997973

Auxin Regulation and MdPIN Expression during Adventitious ... · Auxin and MdPIN S regulate...

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