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Article

Overexpression of OsPIN5b Alters Plant Architecture and Impairs Cold Tolerance in Rice (Oryza sativa L.)

by
Xiaoyu Fu
1,†,
Guo Chen
1,†,
Xinya Ruan
1,
Guozhang Kang
2,
Dianyun Hou
1 and
Huawei Xu
1,*
1
College of Agriculture, Henan University of Science and Technology, Luoyang 471000, China
2
National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450046, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(7), 1026; https://doi.org/10.3390/plants14071026
Submission received: 25 February 2025 / Revised: 17 March 2025 / Accepted: 19 March 2025 / Published: 25 March 2025
(This article belongs to the Special Issue Mitigation Strategies and Tolerance of Plants to Abiotic Stresses—2nd Edition)
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Abstract

Auxin plays a versatile role in regulating plant growth and development. The auxin efflux carrier PIN-FORMED (PIN) proteins dictate the distribution and maximum of auxin within various tissues. Despite extensive research on OsPINs in recent years, their functions in abiotic stress resistance, particularly cold tolerance, remain poorly understood. Here, we investigated the role of OsPIN5b in rice (Oryza sativa L.) growth and development, as well as its contribution to cold tolerance using overexpression technology. Overexpression of OsPIN5b (OE) resulted in reduced shoot height and a lower number of adventitious roots at the seedling stage. Transgenic rice plants exhibited an earlier heading date, stunted growth, and compromised agronomic traits, including shortened panicle length, decreased grain number per panicle, reduced seed size, and lower seed setting rate during the reproductive stage. Auxin content in the transgenic lines was significantly elevated, as indicated by the upregulation of the auxin-responsive gene OsIAA20 and increased auxin levels quantified using a newly developed method. Compared with wild-type plants, the cold tolerance of OE plants was markedly reduced, as evidenced by lower survival rates, higher levels of electrolyte leakage, and increased malondialdehyde (MDA) production following cold treatment. In line with this, the transgenic lines produced less soluble sugar and proline, while accumulating more hydrogen peroxide (H2O2) and superoxide anion radicals ( O 2 − ) after cold treatment. Furthermore, the activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were notably decreased upon cold treatment compared with those in WT plants. Additionally, OsRBOHH, which plays a role in ROS production, was significantly upregulated in transgenic lines both before and after chilling stress, suggesting that OsRBOHH plays a potential role in regulating ROS production. Collectively, overexpression of OsPIN5b substantially disturbs auxin homeostasis, resulting in impaired plant architecture and agronomic traits. More importantly, the upregulation of OsPIN5b compromises rice cold tolerance by perturbing ROS homeostasis and adversely influencing the accumulation of soluble sugar and proline.

1. Introduction

Rice (Oryza sativa L.) is one of the most important crops worldwide, originating from tropical and subtropical regions. Compared to other cereal crops, rice exhibits greater sensitivity to cold stress [1,2,3,4]. Additionally, the increasing frequency of extreme weather events exposes plants to various abiotic stresses, including cold stress. Cold stress not only restricts the geographical distribution of plants but also significantly reduces their productivity [5,6]. For rice, cold stress primarily impairs growth and development during the early stages of seedling establishment [7], as well as at the booting stage [2,8]. Research has demonstrated that low temperatures can reduce rice yields by 30–40% in temperate regions [9]. Therefore, improving rice cold tolerance is essential for ensuring food security [10].
In recent decades, extensive research has been conducted on the molecular regulatory mechanisms underlying plant cold tolerance [11,12,13]. Notably, the well-known C-repeat binding factor (CBF)/dehydration-responsive element binding factor (DREB) genes play crucial roles in regulating plant cold tolerance [14,15,16]. Upregulation of OsDREB1s, such as OsDREB1A, OsDREB1B, OsDREB1F, and OsDREB1G, has been shown to significantly enhance rice cold tolerance [17,18,19,20]. In contrast, several studies have demonstrated that plant cold tolerance is not directly correlated with the expression levels of these genes [21,22,23], highlighting the complexity of the molecular mechanisms underlying plant cold adaptation. Additionally, it has been reported that Ca2+ signaling is involved in regulating plant cold adaptation. For example, COLD1 (CHILLING-TOLERANCE DIVERGENCE 1) and OsCNGC9 (CYCLIC NUCLEOTIDE-GATED CHANNEL 9) regulate rice cold tolerance by facilitating Ca2+ influx in response to cold stress [10]. Cold stress triggers the production of ROS and regulates plant cold tolerance [24,25]. Low levels of ROS, typically produced during the early stages of stress, function as signaling molecules to induce a variety of stress responses, thereby enhancing stress tolerance. In contrast, excessive accumulation of ROS at later stages of stress can cause cellular damage and severely impair plant growth and development [13,23]. However, the precise mechanisms by which plants modulate ROS homeostasis remain unclear. Additionally, numerous regulatory modules, such as SEC13 Homolog 1 (SEH1)-DREB1s-cold regulated (or responsive) genes (CORs), temperature-induced lipocalins (TIL1)-fatty acid desaturases (FADs), NAM, ATAF1/2, and CUC 5 (NAC5)-abscisic acid insensitive 5 (ABI5)-CORs, chilling-tolerance in Geng/japonica rice 3 (COG3)-filamentation temperature-sensitive H 2 (FtsH2)-D1, mitogen-activated protein kinase 6 (MAPK6)-inducer of CBF expression 1 (ICE1)/ideal plant architecture 1 (IPA1), histone deacetylase 716 (HDA716)-basic leucine zipper 46 (bZIP46)-DREB1A/COLD1, and COG1-somatic embryogenesis receptor kinases-like 2 (SERL2)-MAPK3, have been reported to be involved in rice cold tolerance [26,27,28,29,30,31,32].
As the first-discovered phytohormone, auxin not only regulates nearly all aspects of plant growth and development [33,34,35,36] but also plays a crucial role in mediating responses to multiple biotic and abiotic stresses, including cold stress [37,38]. Exogenous application of auxin enhances plant cold tolerance [39], and auxin levels significantly increase in response to cold treatment [40]. This findings suggest that auxin plays a crucial role in regulating plant cold tolerance. The biosynthesis, transport, conjugation, and catabolism of auxin can markedly influence auxin levels and spatial distribution within plants [41,42,43], which may subsequently modulate auxin signaling pathways and thereby affect cold tolerance. However, investigations have demonstrated that plant cold tolerance is likely linked to polar auxin transport (PAT) [44]. PAT is closely associated with auxin transport carriers, specifically the PIN-FORMED (PIN) proteins and influx carriers AUXIN RESISTANT1 (AUX1)/LAX family members [45,46]. Among these, PIN carriers play a particularly crucial role in PAT [47]. An earlier report demonstrated that low temperature can suppress auxin transport rate in plants [48]. Cold treatment inhibits basipetal auxin transport in the inflorescence stem, while room temperature restores it [49]. Cold stress primarily blocks the intracellular trafficking of PIN2 and PIN3, thereby inhibiting shootward auxin transport [44]. GNOM, a SEC7-domain-containing ARF-GEF that affects PAT and development in plants [50,51,52], has been shown to mediate the cold stress response in Arabidopsis [53]. In agreement with this, our studies showed that OsPIN9 positively regulates rice cold tolerance [54,55], whereas the mutation of OsPIN1b impairs rice cold tolerance [56]. Collectively, accumulating evidence suggests that plant cold tolerance is closely associated with PAT, particularly the PIN genes. However, the detailed molecular mechanisms remain largely elusive.
The rice genome contains 12 OsPIN genes, and their tissue-specific expression patterns as well as responses to various hormones and abiotic stresses have been extensively investigated [57,58,59]. OsPIN5b is targeted to endoplasmic reticulum (ER) and plays a crucial role in regulating auxin homeostasis, transport, and distribution, thereby influencing rice plant architecture and yield [60]. However, the role of OsPIN5b in regulating rice cold tolerance remains unclear. In this study, we upregulated the expression of OsPIN5b and found that it regulates rice plant architecture and key agronomic traits, likely due to altered auxin homeostasis. Furthermore, we observed that overexpression of OsPIN5b significantly impairs rice cold tolerance, primarily due to the perturbation of ROS homeostasis and decline in the accumulation of soluble sugar and proline.

2. Results

2.1. Generation of OsPIN5b-Overexpressing Lines and Phenotypes of Transformants

To investigate the role of OsPIN5b in regulating plant growth, development, and abiotic stress resistance, we employed overexpression technology to upregulate its expression. A plasmid harboring the OsPIN5b ORF under the control of a strong constitutive Ubiquitin promoter (pUbi) was introduced into the rice cultivar Nipponbare via Agrobacterium-mediated transformation (Figure 1A). The presence of the transgene in the resulting transgenic lines was confirmed by PCR using genomic DNA as the template. Compared with the WT plants, qRT-PCR analysis revealed that OsPIN5b was greatly elevated in different OE lines (Figure 1B). Among these, lines A3 and A4, which exhibited similar expression levels of OsPIN5b and were subsequently renamed as OE1 and OE2, were employed for further investigation. We then examined the expression of OsPIN5b in leaves, roots, and stem bases of these two transgenic lines. The highest increase in expression was observed in the leaves, followed by the stem bases, while the lowest expression was detected in the roots (Figure 1C). These results indicate that OsPIN5b expression is substantially increased in the transgenic lines.
We subsequently analyzed the phenotype of OE lines at the seedling stage. The plant height, root length, and number of adventitious roots in 7-day-old seedlings were comparable to those in WT plants (Figure S1A). However, as the plants continued to grow, consistent with previous findings on OsPIN5b overexpression [60], upregulation of OsPIN5b resulted in retarded growth in 14-day-old seedlings. Specifically, the shoot height of OE lines was significantly reduced compared with WT plants, and the number of adventitious roots in OE lines was notably lower than that in WT plants (Figure S1B). In contrast, the root length in the transgenic plants remained comparable to that of WT plants (Figure S1B), which differs from previous findings [60]. These results suggest that overexpression of OsPIN5b primarily affects shoot height and adventitious root growth rather than primary root growth, at least during the seedling stage.
Apart from altering the phenotype at the seedling stage, overexpression of OsPIN5b also significantly impaired rice growth and development at the mature stage. The plant height of the OE lines was reduced by approximately 20% compared with WT plants, primarily due to a decrease in internode length. Additionally, we observed that the heading date of OE lines occurred approximately one week earlier than that of WT plants (Figure S2).
In addition, the upregulation of OsPIN5b impaired panicle development and seed setting rate, consistent with previous findings [60]. Specifically, compared to WT plants, OE lines exhibited significant reductions in panicle length, number of branches per panicle, grain number per panicle, grain weight per panicle, and seed setting rate (Figure S3). These results suggest that the overexpression of OsPIN5b adversely affects these key agronomic traits.

2.2. Upregulation of OsPIN5b Disrupts Auxin Homeostasis

To investigate the effect of upregulation of OsPIN5b on auxin homeostasis, we first examined the expression levels of OsYUC and OsPIN genes in both WT and OE roots. Several OsYUC genes, including OsYUC3, OsYUC7, and OsYUC8, displayed a significant decrease in expression in OE lines compared with WT plants, while the expression levels of other OsYUC genes remained comparable to those in WT plants (Figure 2A). Given that OsYUC genes are substantially upregulated under auxin deficiency, which suggests a potential metabolic compensation mechanism for auxin deficiency [61], it is plausible to infer that the depressed expression of OsYUC genes in OE lines may be attributed to elevated auxin levels. Furthermore, most OsPINs remained unchanged in OE lines compared with WT plants, with the exception of OsPIN2 and OsPIN5b (Figure 2B). The high expression of the target gene OsPIN5b is reasonable, as its overexpression primarily leads to the upregulation of OsPIN2, suggesting that these two genes may coordinately regulate auxin transport in rice roots.
Previous studies have demonstrated that OsPIN5b positively regulates free auxin content in rice leaves, roots, and panicles by facilitating the conversion of conjugated auxin forms to free auxin [60]. To investigate the auxin levels in OE lines, we utilized OsIAA20, a gene commonly employed as a marker for evaluating auxin content [62,63,64]. As expected, the expression of OsIAA20 was significantly elevated in both OE leaves and roots (Figure 3A), suggesting that auxin levels likely increased due to the upregulation of OsPIN5b. Furthermore, we employed a newly developed method to measure auxin content in OE lines. Consistent with previous reports, auxin content was notably enhanced in OE leaves and root tips (Figure 3B). These results confirm that OsPIN5b indeed plays a critical role in regulating auxin homeostasis.
In addition, it is documented that the relative ratio of IAA-Asp to IAA decreases upon OsPIN5b upregulation [60]. Given the role of the GH3 family in regulating IAA homeostasis by conjugating amino acid (such as Asp, Ala, and Phe) to indole-3-acetic acid (IAA, the main form of auxin) [65], we further analyzed the expression of several GH3 genes, which display a relative higher expression at the seedling stage [66], in both WT and OE lines. The results showed that most GH3 genes displayed similar expression levels in WT and OE leaves, with the exception of GH3-5 and GH3-7, which were significantly downregulated in OE lines compared with WT plants. Notably, GH3-5 exhibited the highest expression among these genes (Figure S4A), implying that these two genes, particularly GH3-5, are likely involved in regulating auxin levels upon OsPIN5b overexpression. By contrast, several GH3 genes, including GH3-1, GH3-4, GH3-8, and GH3-9, were significantly decreased in OE roots compared to WT roots (Figure S4B), implying that these genes may play a role in regulating auxin homeostasis in OE roots. Additionally, GH3-5, which was upregulated in OE leaves relative to WT plants, exhibited a substantial increase in OE roots. These findings indicate that rice leaves and roots likely possess distinct regulatory mechanisms for auxin homeostasis mediated by GH3 genes.

2.3. Overexpression of OsPIN5b Impairs Rice Cold Tolerance

Previously, we demonstrated that low temperature conditions increase the expression of OsPIN5b [57], implying that OsPIN5b may play a potential role in regulating cold tolerance. To assess the effect of overexpression of OsPIN5b on rice cold tolerance, 14-day-old seedlings were transferred from 30 °C to 4 °C. After cold exposure for 5 days, seedlings were returned to room temperature for an additional 4 days. Approximately half of the WT plants leaves remained normal, whereas nearly all leaves of OE plants displayed rolling and wilting (Figure 4). Survival rate analysis revealed that overexpression of OsPIN5b significantly reduced rice cold tolerance. The survival rate of WT plants was approximately 40%, while it was less than 10% in the OE lines. These results indicate that OE plants are more sensitive to low temperatures compared to WT plants.
Cell death, electrolyte leakage, and malondialdehyde (MDA), were detected to further evaluate the cold tolerance of the OE lines. Trypan blue staining, a well-established method for assessing cell viability and membrane integrity, was employed to evaluate cellular damage. No apparent difference in trypan blue staining was observed between WT and OE leaves before cold stress. However, after 2 days of cold treatment, the intensity of trypan blue staining in OE leaves was notably darker than that in WT plants, particularly in OE2 plants, which exhibited a markedly lower survival rate following recovery (Figure 5A). This suggests that the cell death is more pronounced in the OE lines compared to WT plants. Low temperatures typically cause damage to cell membranes, as evidenced by electrolyte leakage, which is a hallmark of such damage [54,67]. In accordance with trypan blue staining, the electrolyte leakage in OE lines was comparable to that in WT plants under normal conditions but significantly higher than that in WT plants after cold stress treatment (Figure 5B). Consistently, cold stress also led to a marked increase in MDA content in OE lines compared with WT plants (Figure 5C). Collectively, these results strongly suggest that overexpression of OsPIN5b substantially impairs rice cold tolerance.

2.4. Soluble Sugar and Proline Content Decreased in OE Lines After Cold Treatment

Abiotic stresses frequently trigger the accumulation of osmotic regulators, such as soluble sugars and proline, to enhance resistance against environmental stressors [68,69,70]. To evaluate the cold tolerance of OsPIN5b-overexpressing plants, we measured the levels of soluble sugars and proline. Prior to cold treatment, the content of soluble sugars and proline in OE lines was comparable to that in WT plants. However, after 48 h of cold stress, the levels of these osmotic regulators significantly decreased in OE lines compared to those in WT plants (Figure 6). These findings indicate that the lower levels of soluble sugars and proline in OE lines may contribute to decreased cold tolerance following exposure to cold stress.

2.5. Overexpression of OsPIN5b Disturbs ROS Homeostasis

Alongside the well-known CBF/DREB regulon, reactive oxygen species (ROS) also play a vital role in cold stress adaptation [23,54,55]. We then employed diaminobenzidine tetrahydrochloride (DAB 4HCl) staining and NBT staining to detect H2O2 and O 2 − , respectively, in WT and OE leaves before and after cold stress. No obvious difference was observed between WT and OE leaves before cold stress, while after cold stress, the OE leaves accumulated obviously more H2O2 and O 2 − compared to WT plants (Figure 7A,B), indicating that the ROS homeostasis is disrupted in OE lines. ROS homeostasis is tightly associated with antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), which play vital roles in ROS scavenging [13,25]. We then measured these enzyme activities in WT and OE lines before and after cold stress. The activities of these three enzymes in OE lines were comparable to those in WT plants before cold stress. However, following cold stress treatment, the enzyme activities in OE lines were greatly suppressed (Figure 7C). Taken together, these results suggest that overexpression of OsPIN5b disrupts ROS homeostasis in rice, likely due to reduced antioxidant enzyme activities, thereby leading to cellular damage and impaired cold tolerance.

2.6. OsRBOHH Likely Plays a Role in ROS Production in OsPIN5-Overexpressing Lines

Plasma membrane-localized respiratory burst oxidase homologs (RBOH) are crucial for regulating ROS production and play a pivotal role in diverse cellular activities and responses to various abiotic and biotic stresses [71,72,73]. To date, nine OsRBOH genes have been identified in the rice genome [74,75,76]. Given that ROS homeostasis is substantially disrupted in OE lines, we investigated the expression of OsRBOH genes before and after cold stress. Prior to cold stress, only OsRBOHH was significantly upregulated in OE lines compared to WT plants, while the expression of other OsRBOH genes remained largely unchanged (Figure 8A). Upon cold treatment, however, only OsRBOHE showed a downward trend in expression in two OE lines, whereas OsRBOHH still exhibited a significant upregulation in OE lines relative to WT plants (Figure 8B). Considering the pronounced accumulation of ROS in OE lines after cold stress, it is plausible that OsRBOHH plays a vital role in modulating ROS production under cold stress conditions in these lines.

3. Discussion

Low temperature is a critical factor influencing plant growth and development [5,6]. In recent decades, numerous functional genes and regulatory modules have been identified as key players in regulating plant cold tolerance [12,77,78]. Additionally, phytohormones such as brassinosteroids (BR), ethylene, jasmonic acid (JA), abscisic acid (ABA), and auxin also play vital roles in plant cold tolerance [37,38,79]. Although the role of auxin in regulating plant cold tolerance has been established for decades [48], the underlying molecular mechanisms remain largely unknown. In this study, we investigated the function of an endoplasmic reticulum (ER)-localized protein, OsPIN5b, in rice growth and development, as well as its role in regulating cold tolerance through overexpression.
The rice genome possesses 12 OsPIN genes [58,59]. Bioinformatic analysis revealed that three OsPIN genes, namely, OsPIN9, OsPIN10a, and OsPIN10b, are specific to monocots [58]. Previous studies have investigated the roles of several OsPIN genes in regulating rice growth and development. For instance, RNAi-mediated suppression of OsPIN1b expression resulted in increased tiller angles, reduced plant height, and fewer adventitious roots [80]. Further analysis revealed that OsPIN1a and OsPIN1b exhibit redundant functions in rice development, whereas OsPIN1c and OsPIN1d play redundant roles in panicle formation [81]. OsPIN2 is predominantly expressed in the root and shoot base, and functions in determining root system architecture and root gravitropism [82,83,84,85]. The OsPIN5 subfamily comprises three genes: OsPIN5a, OsPIN5b, and OsPIN5c [58]. To date, only OsPIN5b was reported and functions in regulating rice architecture and yield [60]. Overexpression of OsPIN5b increases auxin levels across entire rice plants and impairs rice growth and development. In contrast, suppression of OsPIN5b by RNAi technology reduces auxin levels, promotes rice growth and development, and ultimately improves panicle length and grain yield. These findings suggest the potential significance of OsPIN5b in rice molecular breeding. Based on this finding, another report demonstrated that simultaneous mutation of three genes, OsPIN5b, GS3 (a grain size gene), and OsMYB30 (a cold tolerance gene), using CRISPR/Cas9 technology improves both rice yield and cold tolerance [86], further reinforcing the role of OsPIN5b in regulating rice grain yield. To investigate the role of OsPIN5b, we generated OsPIN5b-overexpressing rice plants (Figure 1). Consistent with previous findings [60], gene expression and auxin content measurements demonstrated that overexpression of OsPIN5b significantly disrupts auxin homeostasis (Figure 2 and Figure 3). Furthermore, OsGH3-5 and OsGH3-7 appear to play a crucial role in regulating this process (Figure 3). However, the mechanism by which the upregulation of OsPIN5b modulates the expression of OsGH3 genes requires further investigation. In addition to altering the architecture at the seedling stage (Figure S1), overexpression of OsPIN5b also impairs panicle development and reduces the seed setting rate (Figure S3), suggesting that auxin homeostasis is critical for regulating rice agronomic traits. Consistently, loss-of-function mutations in the rice YUCCA (YUC) flavin-containing monooxygenase encoding genes OsYUC2 or OsYUC11 result in decreased auxin levels, leading to reduced panicle length, seed setting rate, seed size, and grain weight [87,88]. Additionally, research has shown that increased auxin levels due to mutations in the dioxygenase for auxin oxidation (DAO) gene can cause male sterility [89]. These findings strongly suggest that auxin levels during the reproductive stage must be tightly regulated for normal growth and development. The precise molecular mechanisms underlying auxin homeostasis at this stage warrant further investigation.
Auxin is involved in modulating plant cold tolerance, with polar auxin transport (PAT) likely playing a key role in this process [44]. Given that PIN carriers are crucial for controlling PAT, we hypothesized that PIN genes may be involved in modulating cold tolerance by regulating auxin homeostasis. To test this hypothesis, we investigated the cold tolerance of both wild-type and transgenic lines under cold stress conditions. Our findings indicated that the overexpression of OsPIN5b compromises rice cold tolerance. Compared to WT plants, the survival rate was significantly reduced in OE lines (Figure 4). Consistent with this observation, cell death, electrolyte leakage, and MDA content were markedly increased in OE lines relative to WT plants (Figure 5). Additionally, two critical osmolytes, soluble sugars and proline, were substantially decreased in OE lines (Figure 6). This is consistent with previous research showing that the tomato (Solanum lycopersicum) transcription factor SlWRKY51 enhances cold tolerance by promoting proline accumulation [70], underscoring the importance of proline in low-temperature adaptation. However, the molecular mechanisms underlying the decrease in proline levels in OE lines under low-temperature conditions require further investigation.
Despite the key role of auxin in regulating plant cold adaptation [39], the underlying molecular mechanisms remain largely unknown. Previous reports have demonstrated that OsPIN9 is localized to the plasma membrane and plays a crucial role in regulating auxin distribution in both rice shoots and roots [90]. Our research further revealed that OsPIN9 negatively regulates cold tolerance in rice [54,55]. These results suggest that the spatial distribution of auxin in different tissues likely plays a critical role in regulating rice cold tolerance. By contrast, OsPIN5b is localized to the ER, and modulation of OsPIN5b expression not only alters auxin levels throughout the entire plant by influencing auxin conjugation [60] but also potentially affects the intracellular distribution of auxin, which may contribute to changes in rice cold tolerance (Figure 4). Therefore, it appears that OsPIN5b regulates rice cold tolerance by finely modulating the homeostasis between free auxin and conjugated auxin within cells, rather than by mediating auxin transport between different tissues. Collectively, these results indicate that both the spatial distribution of auxin across different tissues and the homeostasis of free auxin and conjugated auxin within cells likely play pivotal roles in regulating rice cold tolerance. While the molecular mechanism underlying auxin-mediated regulation of plant cold tolerance warrants further investigation.
ROS homeostasis plays a crucial role in regulating plant cold tolerance [23]. Accumulating evidence indicates that excessive ROS production under abiotic stress is a primary factor leading to cellular damage in plants [23,24,91,92,93]. For instance, a point mutation in the low-temperature tolerance 1 (LTT1) gene enhances rice cold tolerance by activating ROS metabolic systems [8]. An APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 negatively regulates rice cold tolerance by suppressing ROS scavenging [94]. In rice, compared to the indica varieties, the japonica varieties generally exhibit higher cold tolerance. This may be attributed to their faster accumulation of ROS during the early cold stage and lower ROS accumulation during recovery stages [23]. Our previous study demonstrated that OsPIN9, a monocot-specific auxin efflux carrier gene in rice negatively regulates cold tolerance, and ROS homeostasis plays a crucial role in the cold response process [54,55]. These findings suggest that auxin is likely linked to ROS regulation and is involved in modulating cold tolerance. In line with this, overexpression of OsPIN5b decreased ROS scavenging ability and resulted in ROS accumulation under cold stress (Figure 7), highlighting the critical role of ROS homeostasis during cold treatments. Additionally, proline plays a crucial role in the antioxidative network that alleviates stress-induced oxidative damage [95]. Our results showed that proline content was significantly reduced in OE lines under cold stress conditions (Figure 6). Consequently, the decreased proline content in OE lines may further contribute to ROS accumulation under low-temperature conditions. Previously, we demonstrated that the mutation of OsPIN9 increased auxin levels in rice leaves, which subsequently triggered rapid ROS accumulation and contributed to enhanced cold tolerance [54]. Conversely, despite overexpression of OsPIN5b also increasing auxin levels (Figure 3), it impaired cold tolerance and ROS scavenging in OE lines (Figure 4 and Figure 7). These results suggest that a distinct regulatory mechanism may govern ROS homeostasis, underscoring the complexity of the molecular processes underlying rice cold adaptation. Additionally, we observed that OsRBOHH was significantly upregulated in OE lines under both normal and low-temperature conditions (Figure 8), indicating its potential role in ROS production in these lines. Given the extremely low expression levels of OsRBOHH in rice roots, leaves, shoots, and calli [74], we suspect that OsRBOHH likely plays a primary role under abiotic stress conditions, particularly under cold stress. However, the mechanism by which OsPIN5b regulates OsRBOHH requires further investigation. To elucidate the genetic relationship between OsPIN5b and OsRBOHH, investigation of OsRBOHH mutation in OsPIN5b-overexpressing lines and assessment of ROS accumulation following cold treatment could potentially provide more conclusive evidence. By contrast, a recent report demonstrated that OsRBOHI is essential for ROS production in rice [96]. However, in our study, the expression levels of OsRBOHI in OE lines were comparable to those in WT plants both before and after cold stress (Figure 8), suggesting that OsRBOHI may play a limited role in ROS production in OE lines.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions and Cold Treatment

The rice japonica variety “Nipponbare” was utilized for the physiological experiments and genetic transformation. Hydroponic experiments were conducted following the method described in our previous study [97]. Briefly, rice seeds were surface-sterilized and subsequently cultured in darkness at 30 °C for 3 to 4 days. The germinated seeds were then transferred to Kimura B complete nutrient solution in plant growth chambers. The chambers were set to maintain a photoperiod of 12 h light (30 °C) and 12 h darkness (25 °C), with a relative humidity of 60–70%.
To evaluate the cold tolerance of rice seedlings, 14-day-old seedlings were transferred to low-temperature conditions (4 °C) for 5 days and then allowed by a recovery period under normal conditions. Subsequently, the survival rate was analyzed.

4.2. Vector Construction and Generation of the Transgenic Plants

PrimeSTAR HS DNA Polymerase (Takara Biotechnology Co., Ltd., Dalian, China) was employed to amplify full-length OsPIN5b from rice cDNA using specific primers listed in Table S1. The plant expression vector pCAMBIA1301-pUbi was kindly provided by Dr. Yao-Guang Liu (College of Life Sciences, South China Agricultural University, Guangzhou, China). The OsPIN5b gene was inserted into pCAMBIA1301-pUbi at the Pst I and BamH I restriction sites. The resulting recombinant vector was introduced into rice variety “Nipponbare” following a previously described protocol [98]. Two homozygous transgenic lines, OE1 and OE2, confirmed by PCR and Hygromycin B screening, were used for further investigation.

4.3. Measurement of Auxin Levels

In addition to gene expression analysis, we employed a recently developed method, with slight modifications, to quantify auxin levels in rice leaves and roots as described in a previous study [99]. Briefly, after a 7-day germination period, approximately 0.05 g of seedling leaves or roots was collected and thoroughly homogenized using a multi-sample tissue grinder (Tiss-Basic48, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) in the presence of liquid nitrogen. Subsequently, 100% ethanol (0.3 mL) was added to the powdered sample, and the mixture was then centrifuged at 12,000 rpm for 10 min at 4 °C. Next, 100 μL of the supernatant was mixed with 900 μL of assay reagent (composed of water, concentrated sulfuric acid, and 0.5 M FeCl3 in a ratio of 25 : 15 : 0.75) and incubated at room temperature for 30 min. The absorbance of the resulting reaction mixture was measured at 540 nm and used for auxin content determination.

4.4. Quantitative Real-Time PCR (qRT-PCR) Analysis

qRT-PCR was performed according to our previously published protocol [57]. At least three biological replicates and three technical replicates were performed to assess gene expression levels. The OsACTIN1 gene (Os03g0718100) served as the internal control. All primers utilized in this study are summarized in Table S1, and the gene names and ID numbers used for qRT-PCR analysis are provided in Table S2.

4.5. Physiological Analysis

Cell death was analyzed using trypan blue staining as previously reported [55,100]. Cell membrane integrity was assessed by measuring electrolyte conductivity and MDA content, following the methods described in a previous report [101]. For the determination of soluble sugar and proline content, 14-day-old seedlings were exposed to low-temperature conditions for 48 h. Subsequently, samples were collected and analyzed using the anthrone method for soluble sugar and the sulfosalicylic acid method for proline in accordance with previously established protocols [102,103]. 3,3′-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining were conducted following previously established protocols [23]. Briefly, rice leaves were incubated in DAB and NBT solutions, respectively, for 24 h at 37 °C. Subsequently, the samples underwent decolorization using 95% ethanol. The activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were measured according to previously described methods [104]. Protein content was quantified using the Coomassie Brilliant Blue G-250 staining method as described previously [105].

4.6. Statistical Analysis

Each experiment was independently replicated at least three times. Statistical analysis of the experimental data was conducted using one-way analysis of variance (ANOVA) via GraphPad Prism version 8.0.2 (GraphPad Software Inc., San Diego, CA, USA). Significance levels were set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). All data are presented as means ± standard deviation (SD).

5. Conclusions

Collectively, in this study, we elucidate the role of OsPIN5b in regulating rice architecture and agronomic traits, with a particular emphasis on its function in modulating cold tolerance. Our findings demonstrate that upregulation of OsPIN5b disrupts auxin homeostasis, which adversely affects plant growth and development. More importantly, upregulation of OsPIN5b significantly impairs rice cold tolerance, likely by suppressing ROS scavenging and reducing levels of soluble sugars and proline. Several issues warrant further investigation. For instance, the mechanism by which the expression levels of OsPIN5b influence auxin conjugation requires elucidation. Additionally, it is important to explore how auxin homeostasis regulates ROS homeostasis. Furthermore, whether OsRBOHH plays a vital role in regulating ROS production under cold stress conditions should be examined. Further investigation into these detailed issues may provide insights into the underlying molecular mechanisms of rice cold tolerance and identify potential targets for breeding cold-tolerant crops.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14071026/s1, Figure S1. Phenotypes of wild-type (WT) and OsPIN5b-overexpressing lines (OE) at 7 days (A) and 14 days (B) post-germination. Bar = 5 cm. Values are means ± standard deviation (SD; n = 30). Data were analyzed by ANOVA and Tukey’s tests at p < 0.05 significant level. *: p < 0.05; **: p < 0.01; ***: p < 0.001; Figure S2. Overexpression of OsPIN5b results in earlier heading date and retarded rice growth. (A) Phenotype at the headting stage. Bar = 10 cm. (B) The internode length analysis between WT and transgenic lines. Bar = 10 cm. Values are means ± standard deviation (SD; n = 18). Data were analyzed by ANOVA and Tukey’s tests at p < 0.05 significant level. *: p < 0.05; ***: p < 0.001; Figure S3. Overexpression of OsPIN5b influences rice agronomic traits. (A) Phenotype of wild-type (WT) and OE panicles. Bar = 4 cm. (B) Panicle length. (C) Branch number per panicle. (D) Grain number per panicle. (E) Grain weight per panicle. (F) Seed setting rate. Values are means ± standard deviation (SD; n = 38). Data were analyzed by ANOVA and Tukey’s tests at p < 0.05 significant level. *: p < 0.05; ***: p < 0.001; Figure S4. Expression analysis of GH3 genes in WT and OE leaves (A) and roots (B). Values are means ± standard deviation (SD; n = 3). Data were analyzed by ANOVA and Tukey’s tests at p < 0.05 significant level. *: p < 0.05; ***: p < 0.001; Table S1: Primers used in this study; Table S2: Gene name and ID numbers used for qRT-PCR in this study.

Author Contributions

H.X. conceived and designed this research; X.F., G.C. and X.R. performed the experiment; X.F., G.C. and D.H. analyzed the data; H.X. wrote the manuscript; D.H., G.K. and X.F. 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 (32472050), the Open Research Fund of the State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources (SKLCUSA-b202402), the Student Research Training Program of Henan University of Science and Technology (2024424), and the Open Research Fund of the National Key Laboratory of Wheat and Maize Crop Science (SKL2021KF03).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

We sincerely thank Chunzhao Zhao (Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) and Guozhang Kang (The State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University) for constructive discussion. The authors are grateful to all lab members for their useful suggestions and encouragement.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Generation of OsPIN5-Overexpressing lines: (A) schematic diagram of pUbi:OsPIN5b; (B) expression analysis of OsPIN5b in different transgenic lines; (C) expression analysis of OsPIN5b in various tissues of selected transgenic lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
Figure 1. Generation of OsPIN5-Overexpressing lines: (A) schematic diagram of pUbi:OsPIN5b; (B) expression analysis of OsPIN5b in different transgenic lines; (C) expression analysis of OsPIN5b in various tissues of selected transgenic lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
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Figure 2. Expression analysis of OsYUC (A) and OsPIN (B) genes in wild-type and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; ***: p < 0.001.
Figure 2. Expression analysis of OsYUC (A) and OsPIN (B) genes in wild-type and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; ***: p < 0.001.
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Figure 3. Upregulation of OsPIN5b increases auxin content in rice (Oryza sativa L.): (A) expression analysis of OsIAA20 in WT and OE lines; (B) determination of auxin content in WT and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 3. Upregulation of OsPIN5b increases auxin content in rice (Oryza sativa L.): (A) expression analysis of OsIAA20 in WT and OE lines; (B) determination of auxin content in WT and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
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Figure 4. Overexpression of OsPIN5b impairs rice cold tolerance. Bar = 5 cm. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
Figure 4. Overexpression of OsPIN5b impairs rice cold tolerance. Bar = 5 cm. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
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Figure 5. Upregulation of OsPIN5b results in cell damage: (A) trypan blue staining (Bar = 1 cm); (B) electrolyte leakage; (C) malondialdehyde (MDA) contents. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
Figure 5. Upregulation of OsPIN5b results in cell damage: (A) trypan blue staining (Bar = 1 cm); (B) electrolyte leakage; (C) malondialdehyde (MDA) contents. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
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Figure 6. Soluble sugar (A) and proline (B) content measurement in WT and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 6. Soluble sugar (A) and proline (B) content measurement in WT and OE lines. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
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Figure 7. Overexpression of OsPIN5b disturbs ROS homeostasis: (A) DAB staining (Bar = 1 cm); (B) NBT staining (Bar = 1 cm); (C) measurement of CAT, POD and SOD activities. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
Figure 7. Overexpression of OsPIN5b disturbs ROS homeostasis: (A) DAB staining (Bar = 1 cm); (B) NBT staining (Bar = 1 cm); (C) measurement of CAT, POD and SOD activities. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. ***: p < 0.001.
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Figure 8. Expression analysis of OsRBOH genes in wild-type (WT) and OsPIN5b-overexpressing (OE) lines before (A) and after (B) cold treatment. The expression level of OsRBOH genes in WT was set as one. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; ***: p < 0.001.
Figure 8. Expression analysis of OsRBOH genes in wild-type (WT) and OsPIN5b-overexpressing (OE) lines before (A) and after (B) cold treatment. The expression level of OsRBOH genes in WT was set as one. Values are means ± standard deviation (SD) (n = 3). Data were analyzed by ANOVA and Tukey’s tests at a p < 0.05 significance level. *: p < 0.05; ***: p < 0.001.
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MDPI and ACS Style

Fu, X.; Chen, G.; Ruan, X.; Kang, G.; Hou, D.; Xu, H. Overexpression of OsPIN5b Alters Plant Architecture and Impairs Cold Tolerance in Rice (Oryza sativa L.). Plants 2025, 14, 1026. https://doi.org/10.3390/plants14071026

AMA Style

Fu X, Chen G, Ruan X, Kang G, Hou D, Xu H. Overexpression of OsPIN5b Alters Plant Architecture and Impairs Cold Tolerance in Rice (Oryza sativa L.). Plants. 2025; 14(7):1026. https://doi.org/10.3390/plants14071026

Chicago/Turabian Style

Fu, Xiaoyu, Guo Chen, Xinya Ruan, Guozhang Kang, Dianyun Hou, and Huawei Xu. 2025. "Overexpression of OsPIN5b Alters Plant Architecture and Impairs Cold Tolerance in Rice (Oryza sativa L.)" Plants 14, no. 7: 1026. https://doi.org/10.3390/plants14071026

APA Style

Fu, X., Chen, G., Ruan, X., Kang, G., Hou, D., & Xu, H. (2025). Overexpression of OsPIN5b Alters Plant Architecture and Impairs Cold Tolerance in Rice (Oryza sativa L.). Plants, 14(7), 1026. https://doi.org/10.3390/plants14071026

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