Next Article in Journal
SARS-CoV-2 and Immunity: Natural Infection Compared with Vaccination
Next Article in Special Issue
The MdAux/IAA2 Transcription Repressor Regulates Cell and Fruit Size in Apple Fruit
Previous Article in Journal
Bio-Based Degradable Poly(ether-ester)s from Melt-Polymerization of Aromatic Ester and Ether Diols
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 16
10.3390/ijms23168965
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism

by
Huihui Wang
,
Qiqi Ouyang
,
Chong Yang
,
Zhuoyan Zhang
,
Dianyun Hou
,
Hao Liu
* and
Huawei Xu
*
College of Agriculture, Henan University of Science and Technology, Luoyang 471000, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(16), 8965; https://doi.org/10.3390/ijms23168965
Submission received: 16 July 2022 / Revised: 8 August 2022 / Accepted: 9 August 2022 / Published: 11 August 2022
(This article belongs to the Special Issue Regulatory Mechanisms of Auxin in Plant Growth and Development)
Browse Figures
Versions Notes

Abstract

:
The distribution and content of auxin within plant tissues affect a variety of important growth and developmental processes. Polar auxin transport (PAT), mainly mediated by auxin influx and efflux transporters, plays a vital role in determining auxin maxima and gradients in plants. The auxin efflux carrier PIN-FORMED (PIN) family is one of the major protein families involved in PAT. Rice (Oryza sativa L.) genome possesses 12 OsPIN genes. However, the detailed functions of OsPIN genes involved in regulating the rice architecture and gravity response are less well understood. In the present study, OsPIN1b was disrupted by CRISPR/Cas9 technology, and its roles in modulating rice architecture and root gravitropism were investigated. Tissue-specific analysis showed that OsPIN1b was mainly expressed in roots, stems and sheaths at the seedling stage, and the transcript abundance was progressively decreased during the seedling stages. Expression of OsPIN1b could be quickly and greatly induced by NAA, indicating that OsPIN1b played a vital role in PAT. IAA homeostasis was disturbed in ospin1b mutants, as evidenced by the changed sensitivity of shoot and root to NAA and NPA treatment, respectively. Mutation of OsPIN1b resulted in pleiotropic phenotypes, including decreased growth of shoots and primary roots, reduced adventitious root number in rice seedlings, as well as shorter and narrower leaves, increased leaf angle, more tiller number and decreased plant height and panicle length at the late developmental stage. Moreover, ospin1b mutants displayed a curly root phenotype cultured with tap water regardless of lighting conditions, while nutrient solution culture could partially rescue the curly root phenotype in light and almost completely abolish this phenotype in darkness, indicating the involvement of the integration of light and nutrient signals in root gravitropism regulation. Additionally, amyloplast sedimentation was impaired in the peripheral tiers of the ospin1b root cap columella cell, while it was not the main contributor to the abnormal root gravitropism. These data suggest that OsPIN1b not only plays a vital role in regulating rice architecture but also functions in regulating root gravitropism by the integration of light and nutrient signals.

1. Introduction

Auxin (mainly indole-3-acetic acid, IAA) is a universal phytohormone in plants, which requires polar transport and regulates various aspects of plant developmental and growth processes, such as embryogenesis, organogenesis, shoot elongation, root development, vascular tissue differentiation and tropisms [1,2]. Auxin is predominantly synthesized in the shoot apex and developing leaf primordia, and then transported to the targeted tissues by a polar auxin transport (PAT) system [3]. PAT is slower and regulated, occurring via carrier-mediated cell-to-cell directional transport [4]. PAT is established and maintained by the auxin transporters in plants: AUXIN/LIKE AUXIN (AUX/LAX) influx carriers, auxin efflux carriers of the PIN-FORMED (PIN) family, ATP-Binding Cassette subfamily B/P-glycoprotein (ABCB/PGP) subfamily of ABC transporters, and the newly described PIN-LIKES (PILS) proteins [2,5,6]. Directionality and rate of auxin movement are mainly controlled by asymmetric localization of the membrane-localized PIN proteins [7,8]. Rice (Oryza sativa L.), the most important staple food of over half of the world’s population [9,10,11], is considered one of the most suitable monocot models for plant molecular biology research because of the establishment of genome databases and mutant resources [12,13]. Extensive progress has been made in understanding the function of AtPIN genes in Arabidopsis [2,14]. However, the functions of OsPIN genes are still largely unknown. PIN1 is the first reported auxin efflux carrier that functions in shoot-basipetal auxin transport in Arabidopsis, and pin1 mutants show a classical PIN-like inflorescence [15,16]. Thereafter, the PIN gene family has been extensively investigated in rice (Oryza sativa L.) [17,18,19], maize (Zea mays L.) [20,21,22], sorghum (Sorghum bicolor) [23], soybean (Glycine max L.) [24,25], cotton (Gossypium hirsutum) [26,27], potato (Solanum tuberosum) [28], Chinese cabbage (Brassica rapa L. ssp. pekinensis) [29], tomato (Solanum lycopersicum) [30], Medicago truncatula [31], Populus [32,33], switchgrass (Panicum virgatum L.) [34], olive (Olea europaea) [35], pear (Pyrus pyrifolia) [36] and coffee (Coffea arabica) [37].
There are 12 PIN genes in the rice genome: four OsPIN1 (OsPIN1a-OsPIN1d), one OsPIN2, three OsPIN5 (OsPIN5a-OsPIN5c), one OsPIN8, and three monocot-specific PIN genes, OsPIN9, OsPIN10a, and OsPIN10b [17,19]. To date, several OsPIN genes have been cloned and functionally characterized, such as the OsPIN1 subfamily [3,7,38], OsPIN2 [39,40,41,42,43,44,45], OsPIN5b [46] and OsPIN10a [47]. A previous study showed that rice OsPIN1b is involved in the regulation of adventitious root emergence and tiller number [3], while further investigation reported that ospin1 single mutants had no dramatic phenotypes and only pin1a pin1b or pin1c pin1d double mutants showed obvious phenotypes, indicating the functional redundancy of OsPIN1 genes in modulating rice architecture [7]. In addition, OsPIN1b is also involved in nitric oxide (NO) or strigolactones (SLs)-induced root elongation under low-nitrogen and -phosphate conditions [38]. OsPIN2 is involved in root elongation growth, root gravitropic responses and lateral root formation patterns via regulating the basipetal auxin flow from the root tip towards the root-elongation zone [42,43,45]. OsPIN5b protein targets the endoplasmic reticulum and is involved in modulating tiller number, root system, panicle length and grain yield in rice [46]. OsPIN9 is highly expressed in the vascular tissue of the root and stem base [19] and plays an important role in modulating adventitious root number and tiller number in rice [48]. OsPIN10a, also designated as OsPIN3t, is mainly expressed in vascular tissue and involved in the regulation of root growth and drought tolerance [47].
Although excellent progress has been made in understanding the underlying mechanisms of OsPIN genes involved in polar auxin transport in recent years, detailed information is still necessary to further dissect the role of OsPIN genes in regulating plant development and response to environmental cues. In this study, we reported the versatile role of OsPIN1b in regulating plant architecture and root gravitropism using ospin1b mutants created by CRISPR/Cas9 technology and further discussed the effects of different cultural conditions on ospin1b root gravitropism.

2. Results

2.1. Tissue-Specific Expression Pattern of OsPIN1b

The tissue-specific expression pattern of OsPIN1b was investigated by qRT-PCR and β-glucuronidase (GUS) assay of transgenic plants expressing an OsPIN1b promoter:GUS (pOsPIN1b:GUS) fusion construct. qRT-PCR analysis showed that OsPIN1b mRNA was transcribed in all tissues tested in 14-day-old seedlings. High levels of OsPIN1b transcript were detected in the sheaths, and moderate levels were detected in the stems and roots, while relatively lower levels were found in the leaves and stem bases (Figure 1A). To confirm the tissue-specific expression of the OsPIN1b gene, the expression pattern of OsPIN1b was assayed by GUS staining. The results show that OsPIN1b is expressed to a relatively high level in early young seedlings and gradually decreases as the plants grow while still keeping a relatively high level in the roots (Figure 1B).

2.2. Induction of OsPIN1b by Exogenous NAA and NPA

1-Naphthaleneacetic acid (NAA) can enter cells passively, and most of the identified OsPIN genes can respond to NAA treatment [7,46,47]. N-1-naphthylphthalamic acid (NPA) is one of the auxin transport inhibitors, which can be employed as a valuable tool to investigate auxin transporter-mediated developmental processes [49].
To test the response of OsPIN1b to NAA or NPA treatment, we monitored the effects of NAA or NPA on the expression of OsPIN1b gene in rice roots by qRT-PCR. The 14-day-old seedlings were exposed to exogenous hormones of NAA at a concentration of 0.1 μM, or NPA at 0.5 μM. The results showed that OsPIN1b responded to exogenous NAA quickly and increased greatly after treatment for 3 h, the expression level was gradually increased, and the highest expression level was detected after treatment for 12 h (Figure 2A). For NPA treatment, with the treatment time increasing from 0 to 9 h, the transcript abundance increased gradually while decreasing after treatment for 12 h (Figure 2A).
To verify whether OsPIN1b is involved in polar auxin transport, 7-days seedlings of the pOsPIN1b:GUS transgenic plants were exposed to 0.1 µM NAA or 0.5 µM NPA, and GUS staining was performed after one-day treatment. Consistent with qRT-PCR results, GUS staining was darker after treatment with NAA or NPA, confirming the induction of OsPIN1b under NAA or NPA treatment (Figure 2B).

2.3. Generation and Identification of Rice ospin1b Mutants

To further investigate the function of OsPIN1b in rice, two ospin1b mutants, designated as ospin1b-1 and ospin1b-2, were generated using CRISPR/Cas9 technology. The ospin1b-1 and ospin1b-2 mutants contained a 1- and 4-bp deletion, respectively, in the first exon of OsPIN1b (Figure 3). The OsPIN1b mutation caused frame-shift mutation and premature termination of translation, and the mutation proteins contained 196 and 195 amino acids in ospin1b-1 and ospin1b-2 mutants, respectively. The function of PIN proteins is closely dependent on the number of transmembrane helices [50]. Bioinformatics analysis showed that only two transmembrane helices were present in the mutated OsPIN1b proteins, while the native OsPIN1b protein contains 10 transmembrane helices (Figure S1). These results indicate that ospin1b mutants lacked OsPIN1b. Off-target analysis confirmed that none of the off-target mutations was introduced into the ospin1b mutants (Figure S2). The progeny of these homozygous mutants was used for further study.

2.4. Auxin Homeostasis Is Disrupted in ospin1b Mutants

Due to the fact that OsPIN1b can respond to NAA or NPA treatment (Figure 1), we further evaluated the response of ospin1b mutants to NAA or NPA treatment. Although the shoot height of ospin1b mutants under NAA treatment showed a similar decrease compared with that under normal conditions, NPA treatment greatly decreased the shoot height of ospin1b mutants (Figure 4A,B), indicating that ospin1b mutant shoots were more sensitive to NPA treatment. The root length of ospin1b mutants was reduced by 22–26% compared with wild-type (WT) plants under NAA treatment, while it decreased by 32–42% compared with WT plants under normal conditions, indicating that ospin1b roots were more tolerant to NAA treatment than WT roots (Figure 4C). Adventitious root development is tightly associated with auxin homeostasis [3,48]. Consistent with this view, adventitious root number was reduced by 20–24% compared to WT plants under normal conditions, while NPA treatment dramatically suppressed the emergence of adventitious roots, which was greatly decreased by 76–77%, in ospin1b mutants compared to that in WT plants (Figure 4D), indicating that ospin1b mutant roots were more sensitive to NPA treatment. Collectively, these data strongly illustrate that auxin homeostasis in ospin1b mutants is disturbed.

2.5. Mutation of OsPIN1b Results in Reduced Growth of Shoot and Primary Root, as well as Decreased Number of Adventitious Roots

To understand the function of OsPIN1b at the seedling stage, the phenotypes of 7-day-old and 14-day-old seedlings were investigated. Compared with WT control, shorter primary roots of the ospin1b mutants were the most obvious phenotype. The primary root length was significantly decreased in comparison with the WT plants even after germination for only 7 days (Figure 5A). After germination for 14 days, the shoot height, root length and adventitious root number in ospin1b mutants were all significantly lower than that in WT plants (Figure 5B). These results show that the mutation of OsPIN1b significantly suppresses shoot and root growth and affects the development of adventitious roots.

2.6. Disruption of OsPIN1b Causes Shorter and Narrower Leaves and Larger Leaf Angle

The 14-day-old seedlings were transferred into the soil and then cultured under normal conditions. Generally, ospin1b mutants grew more slowly than the WT plants, and the leaves of the ospin1b mutants were shorter and narrower than those of the WT plants. As shown in Figure 6A, the flag leaves of the ospin1b mutants were distinguished as shorter than that of the WT plants at the heading stage (90 days after germination), and the flag leaf length of the WT was about 30.5 cm on average, while it was about 25.0 cm in ospin1b mutants. The leaf width of ospin1b was significantly decreased in comparison with WT plants, which was 7–8% narrower than the WT plants (Figure 6A). Additionally, we also noticed that the flag leaf angle of ospin1b was larger than WT plants, which was significantly increased by 37.4% and 74.7% in ospin1b-1 and ospin1b-2 mutants, respectively, compared with WT plants (Figure 6B). These results suggest that OsPIN1b is involved in the regulation of leaf development.

2.7. Loss of function of OsPIN1b Leads to Decreased Plant Height, Increased Tiller Number, Reduced Panicle Length and Increased Grain Yield at the Mature Stage

At the mature stage, the ospin1b mutants exhibited a clear phenotype, including decreased plant height, increased tiller number and shorter panicle compared with WT plants. The plant height of ospin1b mutants was significantly reduced by 6–7%, while the tiller number greatly increased by 47–51% compared with WT plants (Figure 7A). ospin1b mutants also had a decrease in the panicle length of about 20%, leading to a decrease in grain number per panicle relative to control plants of about 32% (Figure 7B). However, the grain yield per plant of ospin1b was significantly increased by 15–17% compared to wild-type plants, which was mainly attributed to the increased tiller number (Figure 7).
To verify which internode is attributed to the decreased plant height in ospin1b mutants, we further examined the lengths of the first to fifth internodes in WT and ospin1b mutants. The first internode length was significantly reduced in ospin1b mutants compared to WT plants, the second to fourth segments showed no significant difference from that of the WT plants, while the fifth internode length was significantly higher than that of the WT (Figure S3). These data suggest that the decreased plant height of ospin1b mutants is mainly caused by the decreased length of the first internode.

2.8. Mutation of OsPIN1b Alters Root Gravitropism

Root gravitropism is closely associated with auxin content and distribution within plant roots [51]. OsPIN1b is mainly expressed in root tips [19] and is probably involved in regulating root gravitropism. As expected, we noticed that ospin1b root tips exhibited a curly phenotype after germination for 3 days cultured in tap water and darkness (Figure 8A). We then further investigated the root phenotype cultured in half-strength Murashige and Skoog (MS)-agar solid medium in the light. As shown in Figure 8B, the roots of WT plants displayed a classical architecture with one primary root and several adventitious roots, while the roots of ospin1b lines lost the gravitropism and exhibited curly phenotype after being cultured for 7 days. These data strongly suggest that the mutation of OsPIN1b severely influences root gravitropism in rice.

2.9. The Root Agravitropism of ospin1b Mutants Is Influenced by Various Culture Conditions

Surprisingly, the curly root phenotype was not observed consistently. Further investigation showed that the curly root phenotype was tightly associated with culture conditions. ospin1b root showed a relatively higher curly root proportion when cultured with tap water or distilled water, especially when cultured with tap water, and almost all mutants displayed curly root phenotype, whereas a relatively lower curly root proportion was observed when cultured with the nutrient solution regardless of lighting conditions (Figure 9), indicating that culture conditions, especially the nutrient status, can indeed influence ospin1b root gravitropism. In addition to the nutrient conditions, lighting conditions also affected ospin1b root gravitropism. In light, about 40% ospin1b roots showed curly root phenotype cultured with nutrient solution (Figure 9A), while darkness almost completely abolished nutrient solution-grown ospin1b curly root phenotype (Figure 9B). In contrast, ospin1b mutant roots showed a relatively higher curly root proportion in the dark compared with that in light when both were cultured with distilled water (Figure 9A,B). These results demonstrate that light is also involved in regulating ospin1b root gravitropism.

2.10. Amyloplast Sedimentation Is Impaired in ospin1b Mutants, While It Is Not the Main Contributor to ospin1b Root Agravitropism

Evidence suggests that root gravitropism is tightly related to the starch content in root tips [52], and the sedimentation of starch-filled plastids (amyloplasts) in columella cells of the root cap is responsible for gravity sensing [53]. To determine whether ospin1b root agravitropism is associated with amyloplast sedimentation, we assayed the amyloplast sedimentation in tap water-grown rice root tips in darkness by light microscopy. Root caps of the WT and ospin1b plants were both stained intensely, and the staining was mainly restricted within columella cells. In contrast, the staining in ospin1b root caps was mainly detected in the inner tiers rather than the peripheral tiers of columella cell (Figure 10A), indicating that the amyloplast sedimentation is impaired in ospin1b root caps.
For further assay as to whether the impaired amyloplast sedimentation in ospin1b root caps caused root agravitropism, we assayed the amyloplast sedimentation in ospin1b roots cultured with the nutrient solution in darkness, which showed a normal root phenotype. Staining results showed that there was no obvious difference in amyloplast sedimentation in nutrient solution-grown roots and tap water-grown roots (Figure 10B), indicating that the impaired amyloplast sedimentation is not the main contributor influencing ospin1b root gravitropism.

2.11. The Curly Root Phenotype in ospin1b Mutants Is Attributed to the Disturbance of Polar Auxin Transport and Intracellular Trafficking of OsPIN Proteins

The asymmetric redistribution of auxin mediated by PIN carriers plays a vital role in modulating root development and gravitropism [53]. To dissect whether the ospin1b root agravitropism is caused by asymmetric redistribution of auxin mediated by PIN distribution and intracellular trafficking, we performed NPA and intracellular trafficking inhibitor fungal toxin brefeldin A (BFA) to treat ospin1b roots. NPA is an effective auxin transporter inhibitor [54], and BFA can specifically suppress vesicle trafficking [55]. Compared to the curly roots in tap water-grown ospin1b mutants, 0.01 μM NPA treatment partially rescued the curly root phenotype in ospin1b mutants (Figure 11A), while 1 μM BFA treatment almost completely abolished ospin1b curly root phenotype (Figure 11B). These results suggest that both the distribution and subcellular trafficking of OsPIN proteins are involved in regulating ospin1b root gravitropism.

3. Discussion

Root development, which is closely associated with environmental parameters and phytohormones, is a fairly complex process. Although excellent progress has been made in root development for the model dicotyledon plant species Arabidopsis thaliana, the monocot model plant rice, characterized by a dense fibrous root system, still needs further dissection [56]. A variety of genes have been identified and functionally characterized in regulating rice root development [57], and almost all phytohormones are involved in modulating rice root development, among which auxin act as a central node to regulate root development communicated with other phytohormones [58]. In this present study, ospin1b mutants created by CRISPR/Cas9 technology were employed to systematically evaluate the phenotypic alteration at different development stages and environmental cues and nutrient status involved in regulating ospin1b root gravitropism were also dissected.
Auxin is necessary for almost all cellular processes, and an appropriate auxin level and distribution within plant tissues is associated with plant architecture determination [59,60]. PIN genes, which play a central role in auxin efflux, are closely related to auxin distribution, and disturbing the expression of PIN genes gives rise to abnormal architecture [61,62]. For example, mutation of AtPIN1 causes the classical needle-like inflorescence in Arabidopsis [63], and disruption of AtPIN4 affects root patterning [64].
The rice genome contains 12 PIN genes [19], and disturbing the expression of almost all OsPIN genes identified so far could impact rice architecture [3,7,39,46,47,48]. Other genes related to auxin synthesis, conjugation, storage, transport and catabolism also play a crucial role in regulating plant architecture [60,65,66,67,68,69]. One of the most important reasons is likely due to the disturbance of auxin homeostasis caused by the changed expression of these genes. A previous report has demonstrated that OsPIN1b is involved in auxin transport in rice [3]. Our results show that OsPIN1b is universally expressed in rice, implying that OsPIN1b might play a versatile role in rice development, especially in root development. We also noticed that OsPIN1b showed a strong expression in the coleoptile at the early seedling stage (Figure 1B), and further studies are needed to understand how OsPIN1b functions in the coleoptile at this developmental stage. Additionally, OsPIN1b can be quickly responsive to NAA and NPA treatment (Figure 2), indicating the involvement of OsPIN1b in auxin homeostasis. Further results showed that ospin1b mutant shoots and roots were both more sensitive to NPA treatment (Figure 4), indicating that the mutation of OsPIN1b disturbs the auxin homeostasis in rice plants. Consistently, OsPIN1b plays a critical role in orchestrating rice architecture, as evidenced by the pleiotropic phenotypes of ospin1b mutants at different development stages (Figure 5, Figure 6, Figure 7 and Figure 8). In addition to the shorter shoots and roots and reduced adventitious root number at the seedling stage (Figure 5), leave length and width, as well as leaf angle, were also changed in ospin1b mutants at the tilling stage (Figure 6). Mutation of GmPIN1 genes also caused a change in leaf petiole angle in soybean (Glycine max) [70], indicating PIN1 genes are likely to be involved in leaf angle regulation in plants. Consistent with the previous study [3], the tiller number of ospin1b mutants was also significantly increased compared with WT plants, and this change was concomitant with shorter panicles and, resultantly, decreased grain number per panicle at the mature stage, while the grain yield of ospin1b still increased significantly compared to WT plants (Figure 7), which was mainly attributed to the increased tiller number. Consistently, down-regulation of TaPIN1s also increased the tiller number and grain yield in wheat (Triticum aestivum) [71], suggesting that PIN1 genes have the potential to regulate tiller number and grain yield in plants. Unexpectedly, we did not observe the larger tiller angle in ospin1b mutants, which was a clear phenotype in the RNAi plants [3]. It has been demonstrated that OsLAZY1, a gravitropism-related gene, regulates rice tiller angle by affecting the asymmetric redistribution of auxin [72,73], and overexpression of another auxin efflux carrier gene, OsPIN2, also increased rice tiller angle, which probably was mediated by suppressing the expression of OsLAZY1 in the shoots [39], indicating tiller angle is closely associated with the expression of auxin transporters [74]. Collectively, the pleiotropic phenotypes of ospin1b mutants are reminiscent of the phenotype of OsIAAGLU-overexpressing transgenic rice, and these transgenic lines also showed decreased plant height, increased tiller number, larger leaf angle and root agravitropism [60].
PIN genes play a vital role in fine-tuning the content and distribution of auxin in various tissues [75,76], and auxin distribution is closely associated with root gravitropism [77]. Therefore, the proper polarization and expression levels of PIN proteins are necessary to maintain normal gravitropism in plants [77]. ospin1b mutants showed a curly root phenotype (Figure 8), indicating root gravitropism is impaired in ospin1b mutants. Similarly, disruption of the expression of OsPIN2 also resulted in a curly root phenotype, and this phenotype could be almost abolished with NPA treatment, indicating that the curly root phenotype is closely related to polar auxin transport [42]. In line with this, we also noticed that NPA or BFA treatment could partially or almost completely abolish the curly root phenotype (Figure 11), emphasizing the involvement of polar auxin transport in modulating ospin1b root gravitropism. A recent report further confirmed that the wavy root phenotype, caused by the loss of function of OsPIN2, indeed resulted from the disrupted polar auxin transport in root tips, which then led to the alteration of auxin levels, auxin distribution and decreased amyloplast sedimentation in columella cells [45].
It was demonstrated that the first two tiers of root columella cells perceive the gravity stimulation [78] and transmit the signal to the zone of cell elongation, and the asymmetric accumulated auxin causes the gravitropic responses [79]. Conversely, the peripheral cap cells do not function in root gravity sensing [78]. In our observation, amyloplast sedimentation was decreased in peripheral tiers of ospin1b columella cells, whereas there was no obvious difference between nutrient solution-grown and tap water-grown ospin1b roots in the dark (Figure 10), indicating that the impaired amyloplast sedimentation in ospin1b root cap is not the main contributor to the curly root phenotype. Considering that OsPIN1b was highly expressed in root cap [19] and could be rapidly induced by NAA (Figure 2), it is reasonable to speculate that OsPIN1b is probably involved in the regulation of root gravity by transporting auxin, and this regulation might be disturbed in ospin1b root tips, which subsequently results in the agravitropism and curly root phenotype.
It was reported that various culture conditions could influence ospin2 root gravitropism [42]. Consistently, we also noticed that culture conditions can substantially influence ospin1b root gravitropism (Figure 9). The percentage of curly root in nutrient solution-grown ospin1b mutants under light conditions was significantly higher than that under dark conditions (Figure 9), indicating that lighting conditions are involved in regulating ospin1b root gravitropism. Given that auxin content in roots mainly mediated by polar auxin transport is closely associated with root gravitropism, we reasoned that light would improve the asymmetrical distribution of auxin in ospin1b roots, which ultimately increases the curly root proportion in nutrient solution-grown ospin1b mutants (Figure 9). In addition to light and dark, nutrient status can dramatically affect ospin1b root gravitropism. Almost all ospin1b showed a curly root phenotype cultured in tap water (Figure 9). By contrast, nutrient solution culture greatly rescued the curly root phenotype in ospin1b mutants, especially in darkness (Figure 9). Increasing evidence corroborated that nutrient status plays a dominant role in shaping root architecture, and auxin plays a central role in this process [80]. Different forms of nitrogen affect root growth and basipetal auxin transport, and this process is mainly mediated by different phosphorylation statuses and subcellular trafficking of PIN2 protein [81]. Moreover, the expression and subcellular trafficking of PIN2 are tightly associated with root gravitropism [53], and OsPIN1b also participates in regulating root elongation under low nitrogen and low phosphate conditions [38]. These results suggest that the expression, phosphorylation status, as well as subcellular trafficking of PIN carriers, which are closely associated with nutrient status, play a key role in regulating auxin transport and, correspondingly, root gravitropism. Collectively, root gravitropism is a much more complex biological process, which at least needs the cooperation of environmental cues and nutrient status to orchestrate different auxin carriers and, resultantly, the proper distribution of auxin in the root.
Taken together, OsPIN1b probably acts synergistically with OsPIN2 in regulating polar auxin transport and root gravitropism under different culture conditions in rice roots. Mutation of OsPIN1b impairs auxin homeostasis in rice roots and leads to the difference of lateral auxin gradient in root tips under diverse nutrient conditions and, resultantly, the differential curly root proportion. How the light and nutrient signals control root gravitropism is largely unknown, and we demonstrate that ospin1b mutants represent an excellent tool to study the action of light and nutrients as a signal molecule to regulate root gravitropism. Further functional studies are needed to determine the relationship between light, nutrients and root gravitropism.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Rice japonica variety Nipponbare was used for the physiological experiments and rice transformation. For hydroponic experiments, the sterilized rice seeds were germinated in darkness for 3–4 days at 30 °C and then transferred to Kimura B complete nutrient solution [82] in plant growth chambers with 12-h-light (30 °C)/12-h-dark (25 °C) photoperiods, and the relative humidity was controlled at 60–70%. The initial pH of the solution was adjusted to 4.5–5.0, and the solution was replaced every 3 days. To evaluate the phenotypic traits at the seedling stage, rice plants were grown in plant growth chambers. To assay the agricultural traits at the mature stage, 14-day-old rice plants were transferred to soil and cultured under normal conditions.

4.2. Vector Construction and Generation of the Transgenic Plants

The design of sgRNA targets, off-target analysis and mutation type identification were performed by the online toolkit CRISPR-GE (http://skl.scau.edu.cn/, accessed on 1 June 2017) [83]. A CRISPR/Cas9 system, CRISPR-RICE, which has been used successfully to create ospin9 mutants, was employed to construct the OsPIN1b-editing recombinant vector [84]. For ligation of the synthesized DNA oligos to CRISPR-RICE vector, a TGTGT adapter and an AAAC adapter were added to the 5′ of the forward target sequence and reverse target sequence, respectively, and then a 4-nt overhang at the 5′ and 3′ ends of the oligo duplex was formed after annealing. CRISPR-RICE was digested with Bsa I firstly, and then the annealed oligos were inserted into CRISPR-RICE. The recombinant vector was designated as CRISPR-OsPIN1b.
The recombinant vector CRISPR-OsPIN1b was then transformed into Agrobacterium tumefaciens (EHA105) and employed for rice transformation according to the previous report [85].

4.3. Mutation Detection and Off-Target Analysis

The positive transgenic plants were firstly screened out by PCR using HPT-specific primers. Then a primer pair was designed to amplify genomic DNA fragments containing the sgRNA targeted sequence. The mutation types were then decoded by direct sequencing of PCR products [86]. Two homozygous ospin1b mutants, ospin1b-1 and ospin1b-2, were identified and used for further research.
For off-target analysis, the potential off-target sites were first analyzed by CRISPR-GE, and the genotype of the targeted mutation regarding the top 3 potential off-target sites was further detected by sequencing the PCR amplicon directly [86].

4.4. GUS Staining

The pOsPIN1b:GUS transgenic rice was used for OsPIN1b tissue-specific analysis and NAA or NPA treatment experiments.
The histochemical detection of GUS activity was performed according to the previous report [87]. Briefly, transgenic plant samples were incubated with GUS reaction solution at 37 °C overnight. Green tissues were treated with ethanol before observation to remove the chlorophyll pigmentation. The stained tissues were photographed using a Nikon D7100 digital camera (Nikon Corporation, Tokyo, Japan).

4.5. Quantitative RT-PCR

qRT-PCR was performed according to our previous report [18]. In brief, total RNA was extracted from the samples of WT and ospin1b mutants using RNAiso Plus (Takara Bio Inc., Beijing, China). DNase I-digested RNAs were used for RT using HiScript III RT. SuperMix for qPCR (Nanjing Vazyme Biotech Company, Ltd., Nanjing, China). The gene-specific primers were designed using the online website INTEGRATED DNA TECHNOLOGIES (https://sg.idtdna.com, accessed on 1 January 2018). AceQ Universal SYBR qPCR Master Mix (Nanjing Vazyme Biotech Company, Ltd., Nanjing, China) and Lightcycle® 96 system were employed for qRT-PCR. Three biological replicates and three technical repetitions were performed to assay the gene expression, and OsACTIN1 gene (Os03g0718100) was used as an internal control. All primers used in this study are given in Table S1.

4.6. Exogenous NAA and NPA Treatment

For analysis of the response of OsPIN1b to NAA and NPA treatment, 14-day-old seedlings were transferred into Kimura B complete nutrient solution containing 0.1 μM NAA or 0.5 μM NPA for 0, 3, 6, 9 and 12 h. Roots were collected and used for qRT-PCR analysis.
For NAA and NPA sensitivity experiments, germinated rice seeds of WT and ospin1b mutants were transferred to Kimura B complete nutrient solution containing 0.01 μM NAA or 0.25 μM NPA and grown for 7 days, and then the shoot height, root length and adventitious root number were assessed. WT and ospin1b mutants cultured under normal conditions were employed as a control in this study.

4.7. Amyloplast Sedimentation Experiment

WT and ospin1b seeds were germinated for 2–3 days in tap water and darkness, and then the root tips were sampled for amyloplast sedimentation observation. To observe the amyloplasts in the columella cells of the root cap, roots were soaked and stained with I2-KI solution (0.33% (w/v) I2 and 0.67% (w/v) KI) for 5 min. Then the root tips were rinsed in chloral hydrate (40 g chloral hydrate was dissolved in 10 mL glycerine and 20 mL ddH2O) for 2–6 min. The root tips were observed under the microscope.

4.8. Phenotypic Analysis and Root Gravitropism Evaluation

Plant height, leaf length and width, and root length were measured using a ruler. Leaf angles between the sheath and flag leaf after germination for 90 days were photographed and measured with ImageJ.
For root gravitropism analysis, WT and ospin1b mutants were cultured in Kimura B complete nutrient solution, tap water or distilled water for 2–3 days in light or darkness. For curly root phenotype observation, the seeds of WT and ospin1b mutants were sowed on the surface of half-strength Murashige and Skoog (MS)-agar solid medium for 7 days in the light.
BFA is a valuable tool for deciphering the mechanisms of the polarization of PIN [88]. BFA treatment was performed to study the effect of intracellular trafficking on ospin1b root gravitropism. Germinated seeds were transferred into tap water containing 1 μM BFA and cultured for 4 days, and then the percentage of the curly root was assayed.
To evaluate the effect of polar auxin transport on ospin1b root gravitropism, germinated seeds were transferred into tap water containing 0.01 μM NPA and cultured for 5 days, and then the curly root proportion was analyzed.

4.9. Data Analysis

All physiological experiments were repeated at least three times with consistent results. Experimental data were statistically analyzed by one-way analysis of variance (ANOVA) method in GraphPad PRISM (8.0.2) at the significance levels of p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), and all data are displayed as means ± SD.

5. Conclusions

Mutation of OsPIN1b causes pleiotropic phenotypes at different developmental stages, and ospin1b root gravitropism is cooperatively regulated by the involvement of lighting conditions and nutrient status.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23168965/s1.

Author Contributions

H.X. and H.L. conceived and designed this research; H.W., Q.O., C.Y. and Z.Z. performed the experiment; D.H., H.L. and H.X. analyzed the data; H.X. wrote the manuscript. H.W., D.H., H.L. and H.X. completed the writing review. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (grant number 3110019), the Natural Science Foundation of Henan province (grant number 182300410012) and the Student Research Training Program of Henan University of Science and Technology (grant number 2022452).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We sincerely thank Huwei Sun (Laboratory of Rice Biology in Henan Province, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University) for the provision of the pOsPIN1b:GUS plants. The authors are grateful to all lab members for their useful suggestions and encouragement.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gho, Y.S.; Song, M.Y.; Bae, D.Y.; Choi, H.; Jung, K.H. Rice PIN auxin efflux carriers modulate the nitrogen response in a changing nitrogen growth environment. Int. J. Mol. Sci. 2021, 22, 3243. [Google Scholar] [CrossRef]
  2. Zhou, J.J.; Luo, J. The PIN-FORMED auxin efflux carriers in plants. Int. J. Mol. Sci. 2018, 19, 2759. [Google Scholar] [CrossRef] [PubMed]
  3. Xu, M.; Zhu, L.; Shou, H.; Wu, P. A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol. 2005, 46, 1674–1681. [Google Scholar] [CrossRef] [PubMed]
  4. Wang, M.; Qiao, J.; Yu, C.; Chen, H.; Sun, C.; Huang, L.; Li, C.; Geisler, M.; Qian, Q.; Jiang, A.; et al. The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminium stress. Plant Cell Environ. 2019, 42, 1125–1138. [Google Scholar] [CrossRef]
  5. Balzan, S.; Johal, G.S.; Carraro, N. The role of auxin transporters in monocots development. Front. Plant Sci. 2014, 5, 1–12. [Google Scholar] [CrossRef] [PubMed]
  6. Geisler, M.A.B.; Donato, M.D.; Hao, P.C. A critical view on ABC transporters and their interacting partners in auxin transport. Plant Cell Physiol. 2017, 58, 1601–1614. [Google Scholar] [CrossRef] [PubMed]
  7. Li, Y.; Zhu, J.; Wu, L.; Shao, Y.; Wu, Y.; Mao, C. Functional divergence of PIN1 paralogous genes in rice. Plant Cell Physiol. 2019, 60, 2720–2732. [Google Scholar] [CrossRef]
  8. Gibson, C.L.; Isley, J.W.; Falbel, T.G.; Mattox, C.T.; Lewis, D.R.; Metcalf, K.E.; Muday, G.K. A conditional mutation in SCD1 reveals linkage between PIN protein trafficking, auxin transport, gravitropism, and lateral root initiation. Front. Plant Sci. 2020, 11, 910. [Google Scholar] [CrossRef] [PubMed]
  9. Chen, R.; Deng, Y.; Ding, Y.; Guo, J.; Qiu, J.; Wang, B.; Wang, C.; Xie, Y.; Zhang, Z.; Chen, J.; et al. Rice functional genomics: Decades’ efforts and roads ahead. Sci. China Life Sci. 2022, 65, 33–92. [Google Scholar] [CrossRef]
  10. Karki, S.; Rizal, G.; Quick, W.P. Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway. Rice 2013, 6, 28. [Google Scholar] [CrossRef]
  11. Biselli, C.; Cavalluzzo, D.; Perrini, R.; Gianinetti, A.; Bagnaresi, P.; Urso, S.; Orasen, G.; Desiderio, F.; Lupotto, E.; Cattivelli, L.; et al. Improvement of marker-based predictability of apparent amylose content in japonica rice through GBSSI allele mining. Rice 2014, 7, 1. [Google Scholar] [CrossRef]
  12. Miyao, A.; Tanaka, K.; Murata, K.; Sawaki, H.; Takeda, S.; Abe, K.; Shinozuka, Y.; Onosato, K.; Hirochika, H. Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 2003, 15, 1771–1780. [Google Scholar] [CrossRef]
  13. Hirochika, H.; Guiderdoni, E.; An, G.; Hsing, Y.I.; Eun, M.Y.; Han, C.D.; Upadhyaya, N.; Ramachandran, S.; Zhang, Q.; Pereira, A.; et al. Rice mutant resources for gene discovery. Plant Mol. Biol. 2004, 54, 325–334. [Google Scholar] [CrossRef] [PubMed]
  14. Paponov, I.A.; Teale, W.D.; Trebar, M.; Blilou, I.; Palme, K. The PIN auxin efflux facilitators: Evolutionary and functional perspectives. Trends Plant Sci. 2005, 10, 170–177. [Google Scholar] [CrossRef] [PubMed]
  15. Adamowski, M.; Friml, J. PIN-dependent auxin transport: Action, regulation, and evolution. Plant Cell 2015, 27, 20–32. [Google Scholar] [CrossRef]
  16. Galweiler, L.; Guan, C.; Muller, A.; Wisman, E.; Mendgen, K.; Yephremov, A.; Palme, K. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 1998, 282, 2226–2230. [Google Scholar] [CrossRef]
  17. Miyashita, Y.; Takasugi, T.; Ito, Y. Identification and expression analysis of PIN genes in rice. Plant Sci. 2010, 178, 424–428. [Google Scholar] [CrossRef]
  18. Xu, H.W.; Zhang, Y.W.; Yang, X.Y.; Wang, H.H.; Hou, D.Y. Tissue specificity and responses to abiotic stresses and hormones of PIN genes in rice. Biologia 2022, 77, 1459–1470. [Google Scholar] [CrossRef]
  19. Wang, J.R.; Hu, H.; Wang, G.H.; Li, J.; Chen, J.Y.; Wu, P. Expression of PIN genes in rice (Oryza sativa L.): Tissue specificity and regulation by hormones. Mol. Plant 2009, 2, 823–831. [Google Scholar] [CrossRef] [PubMed]
  20. Forestan, C.; Farinati, S.; Varotto, S. The maize PIN gene family of auxin transporters. Front. Plant Sci. 2012, 3, 16. [Google Scholar] [CrossRef]
  21. Forestan, C.; Meda, S.; Varotto, S. ZmPIN1-mediated auxin transport is related to cellular differentiation during maize embryogenesis and endosperm development. Plant Physiol. 2010, 152, 1373–1390. [Google Scholar] [CrossRef] [PubMed]
  22. Yue, R.; Tie, S.; Sun, T.; Zhang, L.; Yang, Y.; Qi, J.; Yan, S.; Han, X.; Wang, H.; Shen, C. Genome-wide identification and expression profiling analysis of ZmPIN, ZmPILS, ZmLAX and ZmABCB auxin transporter gene families in maize (Zea mays L.) under various abiotic stresses. PLoS ONE 2015, 10, e0118751. [Google Scholar] [CrossRef]
  23. Shen, C.; Bai, Y.; Wang, S.; Zhang, S.; Wu, Y.; Chen, M.; Jiang, D.; Qi, Y. Expression profile of PIN, AUX/LAX and PGP auxin transporter gene families in Sorghum bicolor under phytohormone and abiotic stress. FEBS J. 2010, 277, 2954–2969. [Google Scholar] [CrossRef]
  24. Liu, Y.; Wei, H. Genome-wide identification and evolution of the PIN-FORMED (PIN) gene family in Glycine max. Genome 2017, 60, 564–571. [Google Scholar] [CrossRef]
  25. Wang, Y.; Chai, C.; Valliyodan, B.; Maupin, C.; Annen, B.; Nguyen, H.T. Genome-wide analysis and expression profiling of the PIN auxin transporter gene family in soybean (Glycine max). BMC Genom. 2015, 16, 951. [Google Scholar] [CrossRef] [PubMed]
  26. Zhang, Y.; He, P.; Yang, Z.; Huang, G.; Wang, L.; Pang, C.; Xiao, H.; Zhao, P.; Yu, J.; Xiao, G. A genome-scale analysis of the PIN gene family reveals its functions in cotton fiber development. Front. Plant Sci. 2017, 8, 461. [Google Scholar] [CrossRef]
  27. He, P.; Zhao, P.; Wang, L.; Zhang, Y.; Wang, X.; Xiao, H.; Yu, J.; Xiao, G. The PIN gene family in cotton (Gossypium hirsutum): Genome-wide identification and gene expression analyses during root development and abiotic stress responses. BMC Genom. 2017, 18, 507. [Google Scholar] [CrossRef] [PubMed]
  28. Roumeliotis, E.; Kloosterman, B.; Oortwijn, M.; Visser, R.G.; Bachem, C.W. The PIN family of proteins in potato and their putative role in tuberization. Front. Plant Sci. 2013, 4, 524. [Google Scholar] [CrossRef] [PubMed]
  29. Gao, L.W.; Lyu, S.W.; Tang, J.; Zhou, D.Y.; Bonnema, G.; Xiao, D.; Hou, X.L.; Zhang, C.W. Genome-wide analysis of auxin transport genes identifies the hormone responsive patterns associated with leafy head formation in Chinese cabbage. Sci. Rep. 2017, 7, 42229. [Google Scholar] [CrossRef] [PubMed]
  30. Pattison, R.J.; Catala, C. Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families. Plant J. 2012, 70, 585–598. [Google Scholar] [CrossRef] [PubMed]
  31. Sanko-Sawczenko, I.; Lotocka, B.; Czarnocka, W. Expression analysis of PIN genes in root tips and nodules of Medicago truncatula. Int. J. Mol. Sci. 2016, 17, 1197. [Google Scholar] [CrossRef] [PubMed]
  32. Carraro, N.; Tisdale-Orr, T.E.; Clouse, R.M.; Knoller, A.S.; Spicer, R. Diversification and expression of the PIN, AUX/LAX, and ABCB families of putative auxin transporters in Populus. Front. Plant Sci. 2012, 3, 17. [Google Scholar] [CrossRef] [PubMed]
  33. Liu, B.; Zhang, J.; Wang, L.; Li, J.; Zheng, H.; Chen, J.; Lu, M. A survey of Populus PIN-FORMED family genes reveals their diversified expression patterns. J. Exp. Bot. 2014, 65, 2437–2448. [Google Scholar] [CrossRef] [PubMed]
  34. Xu, K.J.; Sun, F.L.; Wang, Y.F.; Shi, L.L.; Liu, S.D.; Xi, Y.J. The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass. Genet. Mol. Biol. 2016, 39, 62–72. [Google Scholar] [CrossRef]
  35. Velada, I.; Cardoso, H.; Porfirio, S.; Peixe, A. Expression profile of PIN-formed auxin efflux carrier genes during IBA-induced in vitro adventitious rooting in Olea europaea L. Plants 2020, 9, 185. [Google Scholar] [CrossRef]
  36. Qi, L.; Chen, L.; Wang, C.; Zhang, S.; Yang, Y.; Liu, J.; Li, D.; Song, J.; Wang, R. Characterization of the auxin efflux transporter PIN proteins in Pear. Plants 2020, 9, 349. [Google Scholar] [CrossRef]
  37. Huang, X.; Bai, X.; Guo, T.; Xie, Z.; Laimer, M.; Du, D.; Gbokie, T., Jr.; Zhang, Z.; He, C.; Lu, Y.; et al. Genome-wide analysis of the PIN auxin efflux carrier gene family in coffee. Plants 2020, 9, 1061. [Google Scholar] [CrossRef] [PubMed]
  38. Sun, H.; Tao, J.; Bi, Y.; Hou, M.; Lou, J.; Chen, X.; Zhang, X.; Luo, L.; Xie, X.; Yoneyama, K.; et al. OsPIN1b is involved in rice seminal root elongation by regulating root apical meristem activity in response to low nitrogen and phosphate. Sci. Rep. 2018, 8, 13014. [Google Scholar] [CrossRef] [PubMed]
  39. Chen, Y.; Fan, X.; Song, W.; Zhang, Y.; Xu, G. Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnol. J. 2012, 10, 139–149. [Google Scholar] [CrossRef]
  40. Wu, D.; Shen, H.; Yokawa, K.; Baluska, F. Alleviation of aluminium-induced cell rigidity by overexpression of OsPIN2 in rice roots. J. Exp. Bot. 2014, 65, 5305–5315. [Google Scholar] [CrossRef] [PubMed]
  41. Wu, D.; Shen, H.; Yokawa, K.; Baluska, F. Overexpressing OsPIN2 enhances aluminium internalization by elevating vesicular trafficking in rice root apex. J. Exp. Bot. 2015, 66, 6791–6801. [Google Scholar] [CrossRef] [PubMed]
  42. Inahashi, H.; Shelley, I.J.; Yamauchi, T.; Nishiuchi, S.; Takahashi-Nosaka, M.; Matsunami, M.; Ogawa, A.; Noda, Y.; Inukai, Y. OsPIN2, which encodes a member of the auxin efflux carrier proteins, is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice. Physiol. Plant 2018, 164, 216–225. [Google Scholar] [CrossRef] [PubMed]
  43. Wang, L.; Guo, M.; Li, Y.; Ruan, W.; Mo, X.; Wu, Z.; Sturrock, C.J.; Yu, H.; Lu, C.; Peng, J.; et al. LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice. J. Exp. Bot. 2018, 69, 385–397. [Google Scholar] [CrossRef] [PubMed]
  44. Sun, H.; Guo, X.; Xu, F.; Wu, D.; Zhang, X.; Lou, M.; Luo, F.; Xu, G.; Zhang, Y. Overexpression of OsPIN2 regulates root growth and formation in response to phosphate deficiency in rice. Int. J. Mol. Sci. 2019, 20, 5144. [Google Scholar] [CrossRef]
  45. Li, W.Q.; Zhang, M.J.; Qiao, L.; Chen, Y.B.; Zhang, D.P.; Jing, X.Q.; Gan, P.F.; Huang, Y.B.; Gao, J.R.; Liu, W.T.; et al. Characterization of wavy root 1, an agravitropism allele, reveals the functions of OsPIN2 in fine regulation of auxin transport and distribution and in ABA biosynthesis and response in rice (Oryza sativa L.). Crop J. 2022, 10, 980–992. [Google Scholar] [CrossRef]
  46. Lu, G.; Coneva, V.; Casaretto, J.A.; Ying, S.; Mahmood, K.; Liu, F.; Nambara, E.; Bi, Y.M.; Rothstein, S.J. OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis, transport and distribution. Plant J. 2015, 83, 913–925. [Google Scholar] [CrossRef]
  47. Zhang, Q.; Li, J.; Zhang, W.; Yan, S.; Wang, R.; Zhao, J.; Li, Y.; Qi, Z.; Sun, Z.; Zhu, Z. The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance. Plant J. 2012, 72, 805–816. [Google Scholar] [CrossRef]
  48. Hou, M.; Luo, F.; Wu, D.; Zhang, X.; Lou, M.; Shen, D.; Yan, M.; Mao, C.; Fan, X.; Xu, G.; et al. OsPIN9, an auxin efflux carrier, is required for the regulation of rice tiller bud outgrowth by ammonium. New Phytol. 2021, 229, 935–949. [Google Scholar] [CrossRef] [PubMed]
  49. Barbez, E.; Lankova, M.; Parezova, M.; Maizel, A.; Zazimalova, E.; Petrasek, J.; Friml, J.; Kleine-Vehn, J. Single-cell-based system to monitor carrier driven cellular auxin homeostasis. BMC Plant Biol. 2013, 13, 20. [Google Scholar] [CrossRef]
  50. Bennett, T.; Brockington, S.F.; Rothfels, C.; Graham, S.W.; Stevenson, D.; Kutchan, T.; Rolf, M.; Thomas, P.; Wong, G.K.; Leyser, O.; et al. Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure. Mol. Biol. Evol. 2014, 31, 2042–2060. [Google Scholar] [CrossRef] [PubMed]
  51. Marchant, A.; Kargul, J.; May, S.T.; Muller, P.; Delbarre, A.; Perrot-Rechenmann, C.; Bennett, M.J. AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J. 1999, 18, 2066–2073. [Google Scholar] [CrossRef]
  52. Kiss, J.Z.; Wright, J.B.; Caspar, T. Gravitropism in roots of intermediate-starch mutants of Arabidopsis. Physiol. Plant 1996, 97, 237–244. [Google Scholar] [CrossRef]
  53. Su, S.H.; Gibbs, N.M.; Jancewicz, A.L.; Masson, P.H. Molecular mechanisms of root gravitropism. Curr. Biol. 2017, 27, R964–R972. [Google Scholar] [CrossRef]
  54. Abas, L.; Kolb, M.; Stadlmann, J.; Janacek, D.P.; Lukic, K.; Schwechheimer, C.; Sazanov, L.A.; Mach, L.; Friml, J.; Hammes, U.Z. Naphthylphthalamic acid associates with and inhibits PIN auxin transporters. Proc. Natl. Acad. Sci. USA 2021, 118, e2020857118. [Google Scholar] [CrossRef]
  55. Liu, S.; Wang, J.; Wang, L.; Wang, X.; Xue, Y.; Wu, P.; Shou, H. Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family. Cell Res. 2009, 19, 1110–1119. [Google Scholar] [CrossRef]
  56. Coudert, Y.; Perin, C.; Courtois, B.; Khong, N.G.; Gantet, P. Genetic control of root development in rice, the model cereal. Trends Plant Sci. 2010, 15, 219–226. [Google Scholar] [CrossRef] [PubMed]
  57. Mai, C.D.; Phung, N.T.; To, H.T.; Gonin, M.; Hoang, G.T.; Nguyen, K.L.; Do, V.N.; Courtois, B.; Gantet, P. Genes controlling root development in rice. Rice 2014, 7, 30. [Google Scholar] [CrossRef] [PubMed]
  58. Saini, S.; Sharma, I.; Kaur, N.; Pati, P.K. Auxin: A master regulator in plant root development. Plant Cell Rep. 2013, 32, 741–757. [Google Scholar] [CrossRef]
  59. Olatunji, D.; Geelen, D.; Verstraeten, I. Control of endogenous auxin levels in plant root development. Int. J. Mol. Sci. 2017, 18, 2587. [Google Scholar] [CrossRef] [PubMed]
  60. Yu, X.L.; Wang, H.Y.; Leung, D.W.M.; He, Z.D.; Zhang, J.J.; Peng, X.X.; Liu, E.E. Overexpression of OsIAAGLU reveals a role for IAA-glucose conjugation in modulating rice plant architecture. Plant Cell Rep. 2019, 38, 731–739. [Google Scholar] [CrossRef]
  61. Michniewicz, M.; Brewer, P.B.; Friml, J.I. Polar auxin transport and asymmetric auxin distribution. Arab. Book 2007, 5, e0108. [Google Scholar]
  62. Friml, J. Auxin transport-shaping the plant. Curr. Opin. Plant Biol. 2003, 6, 7–12. [Google Scholar] [CrossRef]
  63. Okada, K.; Ueda, J.; Komaki, M.K.; Bell, C.J.; Shimura, Y. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 1991, 3, 677–684. [Google Scholar] [CrossRef]
  64. Friml, J.; Benkova, E.; Blilou, I.; Wisniewska, J.; Hamann, T.; Ljung, K.; Woody, S.; Sandberg, G.; Scheres, B.; Jurgens, G.; et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell 2002, 108, 661–673. [Google Scholar] [CrossRef]
  65. Korasick, D.A.; Enders, T.A.; Strader, L.C. Auxin biosynthesis and storage forms. J. Exp. Bot. 2013, 64, 2541–2555. [Google Scholar] [CrossRef]
  66. Ljung, K. Auxin metabolism and homeostasis during plant development. Development 2013, 140, 943–950. [Google Scholar] [CrossRef]
  67. Damodaran, S.; Strader, L.C. Indole 3-butyric acid metabolism and transport in Arabidopsis thaliana. Front. Plant Sci. 2019, 10, 851. [Google Scholar] [CrossRef]
  68. Mellor, N.; Band, L.R.; Pencik, A.; Novak, O.; Rashed, A.; Holman, T.; Wilson, M.H.; Voss, U.; Bishopp, A.; King, J.R.; et al. Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis. Proc. Natl. Acad. Sci. USA 2016, 113, 11022–11027. [Google Scholar] [CrossRef]
  69. Du, H.; Wu, N.; Fu, J.; Wang, S.; Li, X.; Xiao, J.; Xiong, L. A GH3 family member, OsGH3-2, modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice. J. Exp. Bot. 2012, 63, 6467–6480. [Google Scholar] [CrossRef]
  70. Zhang, Z.Q.; Gao, L.; Ke, M.Y.; Gao, Z.; Tu, T.L.; Huang, L.M.; Chen, J.M.; Guan, Y.F.; Huang, X.; Chen, X. GmPIN1-mediated auxin asymmetry regulates leaf petiole angle and plant architecture in soybean. J. Integr. Plant Biol. 2022, 64, 1325–1338. [Google Scholar] [CrossRef] [PubMed]
  71. Yao, F.Q.; Li, X.H.; Wang, H.; Song, Y.N.; Li, Z.Q.; Li, X.G.; Gao, X.Q.; Zhang, X.S.; Bie, X.M. Down-expression of TaPIN1s Increases the Tiller Number and Grain Yield in Wheat. BMC Plant Biol. 2021, 21, 443. [Google Scholar] [CrossRef] [PubMed]
  72. Li, P.; Wang, Y.; Qian, Q.; Fu, Z.; Wang, M.; Zeng, D.; Li, B.; Wang, X.; Li, J. LAZY1 controls rice shoot gravitropism through regulating polar auxin transport. Cell Res. 2007, 17, 402–410. [Google Scholar] [CrossRef] [PubMed]
  73. Yoshihara, T.; Iino, M. Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways. Plant Cell Physiol. 2007, 48, 678–688. [Google Scholar] [CrossRef]
  74. Zhu, M.; Hu, Y.; Tong, A.; Yan, B.; Lv, Y.; Wang, S.; Ma, W.; Cui, Z.; Wang, X. LAZY1 controls tiller angle and shoot gravitropism by regulating the expression of auxin transporters and signaling factors in rice. Plant Cell Physiol. 2021, 61, 2111–2125. [Google Scholar] [CrossRef] [PubMed]
  75. Robert, H.S.; Offringa, R. Regulation of auxin transport polarity by AGC kinases. Curr. Opin. Plant Biol. 2008, 11, 495–502. [Google Scholar] [CrossRef] [PubMed]
  76. Kleine-Vehn, J.; Friml, J. Polar targeting and endocytic recycling in auxin-dependent plant development. Annu. Rev. Cell Dev. Biol. 2008, 24, 447–473. [Google Scholar] [CrossRef] [PubMed]
  77. Rahman, A.; Takahashi, M.; Shibasaki, K.; Wu, S.; Inaba, T.; Tsurumi, S.; Baskin, T.I. Gravitropism of Arabidopsis thaliana roots requires the polarization of PIN2 toward the root tip in meristematic cortical cells. Plant Cell 2010, 22, 1762–1776. [Google Scholar] [CrossRef]
  78. Blancaflor, E.B.; Fasano, J.M.; Gilroy, S. Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity. Plant Physiol. 1998, 116, 213–222. [Google Scholar] [CrossRef]
  79. Mishra, B.S.; Singh, M.; Aggrawal, P.; Laxmi, A. Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development. PLoS ONE 2009, 4, e4502. [Google Scholar] [CrossRef]
  80. Liu, Y.; von Wiren, N. Integration of nutrient and water availabilities via auxin into the root developmental program. Curr. Opin. Plant Biol. 2022, 65, 102117. [Google Scholar] [CrossRef]
  81. Otvos, K.; Marconi, M.; Vega, A.; O’Brien, J.; Johnson, A.; Abualia, R.; Antonielli, L.; Montesinos, J.C.; Zhang, Y.; Tan, S.; et al. Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport. EMBO J. 2021, 40, e106862. [Google Scholar] [CrossRef] [PubMed]
  82. Xu, H.W.; Ji, X.M.; He, Z.H.; Shi, W.P.; Zhu, G.H.; Niu, J.K.; Li, B.S.; Peng, X.X. Oxalate accumulation and regulation is independent of glycolate oxidase in rice leaves. J. Exp. Bot. 2006, 57, 1899–1908. [Google Scholar] [CrossRef] [PubMed]
  83. Liu, W.; Xie, X.; Ma, X.; Li, J.; Chen, J.; Liu, Y.G. DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol. Plant 2015, 8, 1431–1433. [Google Scholar] [CrossRef]
  84. Wu, S.Y.; Yang, X.Y.; Zhang, Y.W.; Hou, D.Y.; Xu, H.W. Generation of ospin9 mutants in rice by CRISPR/Cas9 genome editing technology. Sci. Agric. Sin 2021, 54, 3805–3817. [Google Scholar]
  85. Hiei, Y.; Ohta, S.; Komari, T.; Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6, 271–282. [Google Scholar] [CrossRef] [PubMed]
  86. Ma, X.; Chen, L.; Zhu, Q.; Chen, Y.; Liu, Y.G. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol. Plant 2015, 8, 1285–1287. [Google Scholar] [CrossRef]
  87. Jefferson, R.A.; Kavanagh, T.A.; Bevan, M.W. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987, 6, 3901–3907. [Google Scholar] [CrossRef]
  88. Kleine-Vehn, J.; Huang, F.; Naramoto, S.; Zhang, J.; Michniewicz, M.; Offringa, R.; Friml, J. PIN auxin efflux carrier polarity is regulated by PINOID kinase-mediated recruitment into GNOM-independent trafficking in Arabidopsis. Plant Cell 2009, 21, 3839–3849. [Google Scholar] [CrossRef]
Ijms 23 08965 g001 550
Figure 1. OsPIN1b expression in different tissues. (A) Tissue-specific expression pattern revealed by qRT–PCR. (B) GUS staining experiments in OsPIN1b promoter: GUS (pOsPIN1b:GUS) transgenic plants. Bar = 2 cm.
Figure 1. OsPIN1b expression in different tissues. (A) Tissue-specific expression pattern revealed by qRT–PCR. (B) GUS staining experiments in OsPIN1b promoter: GUS (pOsPIN1b:GUS) transgenic plants. Bar = 2 cm.
Ijms 23 08965 g001
Ijms 23 08965 g002 550
Figure 2. Expression analysis of OsPIN1b response to NAA and NPA treatment. (A) qRT-PCR analysis of OsPIN1b expression in rice seedling roots treated with NAA or NPA. (B) GUS staining experiment was performed after NAA or NPA treatment for 1 day. Bar = 0.5 cm.
Figure 2. Expression analysis of OsPIN1b response to NAA and NPA treatment. (A) qRT-PCR analysis of OsPIN1b expression in rice seedling roots treated with NAA or NPA. (B) GUS staining experiment was performed after NAA or NPA treatment for 1 day. Bar = 0.5 cm.
Ijms 23 08965 g002
Ijms 23 08965 g003 550
Figure 3. Schematic diagram of OsPIN1b gene mutation sites. (A) Gene structure of OsPIN1b and the sequence of the sgRNA target site. (B) A total of 1- and 4-bp nucleotides were deleted in the ospin1b-1 and ospin1b-2, respectively. The red arrow shows the mutation site.
Figure 3. Schematic diagram of OsPIN1b gene mutation sites. (A) Gene structure of OsPIN1b and the sequence of the sgRNA target site. (B) A total of 1- and 4-bp nucleotides were deleted in the ospin1b-1 and ospin1b-2, respectively. The red arrow shows the mutation site.
Ijms 23 08965 g003
Ijms 23 08965 g004 550
Figure 4. Response of ospin1b mutants to NAA or NPA treatment. (A) Phenotypes of wild-type (WT) and ospin1b mutants under normal, NAA or NPA treatments. Bar = 4 cm. (B) Statistical analysis of shoot height (B), root length (C) and adventitious root number (C) in WT and ospin1b mutants under normal, NAA or NPA treatments. Values are means ± standard deviation (D) (n = 24). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 4. Response of ospin1b mutants to NAA or NPA treatment. (A) Phenotypes of wild-type (WT) and ospin1b mutants under normal, NAA or NPA treatments. Bar = 4 cm. (B) Statistical analysis of shoot height (B), root length (C) and adventitious root number (C) in WT and ospin1b mutants under normal, NAA or NPA treatments. Values are means ± standard deviation (D) (n = 24). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Ijms 23 08965 g004
Ijms 23 08965 g005 550
Figure 5. Phenotypes of wild-type (WT), ospin1b-1 and ospin1b-2 seedlings grown in the nutrient solution. (A) Phenotypes of 7-day-old seedlings. (B) Phenotypes of 14-day-old seedlings. Bar = 4 cm. Values are means ± standard deviation (SD) (n = 16). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 5. Phenotypes of wild-type (WT), ospin1b-1 and ospin1b-2 seedlings grown in the nutrient solution. (A) Phenotypes of 7-day-old seedlings. (B) Phenotypes of 14-day-old seedlings. Bar = 4 cm. Values are means ± standard deviation (SD) (n = 16). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Ijms 23 08965 g005
Ijms 23 08965 g006 550
Figure 6. The phenotype of leaves in wild-type (WT) and ospin1b mutants at the heading stage. (A) Leaf length and width. Bar = 4 cm. (B) Leaf angle. Bar = 1 cm. Values are means ± standard deviation (SD) (n = 16). Data were analyzed by ANOVA and Tukey’s tests. ***: p < 0.001.
Figure 6. The phenotype of leaves in wild-type (WT) and ospin1b mutants at the heading stage. (A) Leaf length and width. Bar = 4 cm. (B) Leaf angle. Bar = 1 cm. Values are means ± standard deviation (SD) (n = 16). Data were analyzed by ANOVA and Tukey’s tests. ***: p < 0.001.
Ijms 23 08965 g006
Ijms 23 08965 g007 550
Figure 7. Phenotypes of wild-type (WT), ospin1b-1 and ospin1b-2 at the mature stage. (A) Plant height and tiller number. The plants were grown in the field and transferred to different plastic pots for photo shoots. Bar = 10 cm. (B) Panicle length, grain number per plant and grain yield per plant. Bar = 5 cm. Values are means ± standard deviation (SD) (n = 24). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 7. Phenotypes of wild-type (WT), ospin1b-1 and ospin1b-2 at the mature stage. (A) Plant height and tiller number. The plants were grown in the field and transferred to different plastic pots for photo shoots. Bar = 10 cm. (B) Panicle length, grain number per plant and grain yield per plant. Bar = 5 cm. Values are means ± standard deviation (SD) (n = 24). Data were analyzed by ANOVA and Tukey’s tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Ijms 23 08965 g007
Ijms 23 08965 g008 550
Figure 8. Mutation of OsPIN1b resulted in root agravitropism in ospin1b mutants. (A) Rice plants were cultured for 3 days with tap water in the dark. (B) Rice plants were cultured for 7 days with half-strength Murashige and Skoog (MS)-agar solid medium in light. Bar = 1 cm.
Figure 8. Mutation of OsPIN1b resulted in root agravitropism in ospin1b mutants. (A) Rice plants were cultured for 3 days with tap water in the dark. (B) Rice plants were cultured for 7 days with half-strength Murashige and Skoog (MS)-agar solid medium in light. Bar = 1 cm.
Ijms 23 08965 g008
Ijms 23 08965 g009 550
Figure 9. Root phenotype of wild-type (WT) and ospin1b mutants cultured under diverse conditions. (A) Rice plants were cultured with nutrient solution, tap water or distilled water in light. (B) Rice plants were cultured with nutrient solution, tap water or distilled water in the dark. Bar = 2 cm. Values are means ± standard deviation (SD) (n ≥ 36). Data were analyzed by ANOVA and Tukey’s tests. ***: p < 0.001.
Figure 9. Root phenotype of wild-type (WT) and ospin1b mutants cultured under diverse conditions. (A) Rice plants were cultured with nutrient solution, tap water or distilled water in light. (B) Rice plants were cultured with nutrient solution, tap water or distilled water in the dark. Bar = 2 cm. Values are means ± standard deviation (SD) (n ≥ 36). Data were analyzed by ANOVA and Tukey’s tests. ***: p < 0.001.
Ijms 23 08965 g009
Ijms 23 08965 g010 550
Figure 10. Amyloplast sedimentation analysis in wild-type (WT) and ospin1b root caps. (A) Rice plants were cultured with tap water in the dark. (B) Rice plants were cultured with a nutrient solution in the dark. The red arrow indicates the peripheral tiers of the root cap columella cell. Bar = 0.1 mm.
Figure 10. Amyloplast sedimentation analysis in wild-type (WT) and ospin1b root caps. (A) Rice plants were cultured with tap water in the dark. (B) Rice plants were cultured with a nutrient solution in the dark. The red arrow indicates the peripheral tiers of the root cap columella cell. Bar = 0.1 mm.
Ijms 23 08965 g010
Ijms 23 08965 g011 550
Figure 11. NPA and BFA treatment substantially rescued ospin1b curly root phenotype. (A) 0.01 μM NPA treatment. (B) 1 μM BFA treatment. Bar = 2 cm.
Figure 11. NPA and BFA treatment substantially rescued ospin1b curly root phenotype. (A) 0.01 μM NPA treatment. (B) 1 μM BFA treatment. Bar = 2 cm.
Ijms 23 08965 g011
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Wang, H.; Ouyang, Q.; Yang, C.; Zhang, Z.; Hou, D.; Liu, H.; Xu, H. Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism. Int. J. Mol. Sci. 2022, 23, 8965. https://doi.org/10.3390/ijms23168965

AMA Style

Wang H, Ouyang Q, Yang C, Zhang Z, Hou D, Liu H, Xu H. Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism. International Journal of Molecular Sciences. 2022; 23(16):8965. https://doi.org/10.3390/ijms23168965

Chicago/Turabian Style

Wang, Huihui, Qiqi Ouyang, Chong Yang, Zhuoyan Zhang, Dianyun Hou, Hao Liu, and Huawei Xu. 2022. "Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism" International Journal of Molecular Sciences 23, no. 16: 8965. https://doi.org/10.3390/ijms23168965

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop