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PIN3 positively regulates the late initiation of ovule primordia in Arabidopsis thaliana

['Li-Qin Hu', 'School Of Life Sciences', 'Biotechnology', 'The Joint International Research Laboratory Of Metabolic', 'Developmental Sciences', 'Shanghai Collaborative Innovation Center Of Agri-Seeds Joint Center For Single Cell Biology', 'Shanghai Jiao Tong University', 'Shanghai', 'Jin-Hui Chang', 'Shi-Xia Yu']

Date: 2022-05

Ovule initiation determines the maximum ovule number and has great impact on seed number and yield. However, the regulation of ovule initiation remains largely elusive. We previously reported that most of the ovule primordia initiate asynchronously at floral stage 9 and PINFORMED1 (PIN1) polarization and auxin distribution contributed to this process. Here, we further demonstrate that a small amount of ovule primordia initiate at floral stage 10 when the existing ovules initiated at floral stage 9 start to differentiate. Genetic analysis revealed that the absence of PIN3 function leads to the reduction in pistil size and the lack of late-initiated ovules, suggesting PIN3 promotes the late ovule initiation process and pistil growth. Physiological analysis illustrated that, unlike picloram, exogenous application of NAA can’t restore these defective phenotypes, implying that PIN3-mediated polar auxin transport is required for the late ovule initiation and pistil length. qRT-PCR results indicated that the expression of SEEDSTICK (STK) is up-regulated under auxin analogues treatment while is down-regulated in pin3 mutants. Meanwhile, overexpressing STK rescues pin3 phenotypes, suggesting STK participates in PIN3-mediated late ovule initiation possibly by promoting pistil growth. Furthermore, brassinosteroid influences the late ovule initiation through positively regulating PIN3 expression. Collectively, this study demonstrates that PIN3 promotes the late ovule initiation and contributes to the extra ovule number. Our results give important clues for increasing seed number and yield of cruciferous and leguminous crops.

Ovule is the precursor of seed. Ovule initiation determines maximum ovule number and has great impact on seed number and yield. Understanding the process and regulation of ovule initiation has both scientific significance and potential application. Although the process and its related genes have been reported, the underling mechanisms remains elusive. We previously reported the asynchronous ovule initiation and the hormones integration in regulating ovule initiation. In this study, we found there is late ovule initiation at floral stage 10 in the wild-type Arabidopsis when the existing ovules initiated at floral stage 9 start to differentiate, which contributes to around 10% extra ovule number. We identified pin3 mutants lacking late-initiated ovules, indicating PIN3 facilitates the late ovule initiation. Exogenous application of auxin analogues revealed that polar auxin transport mediated by PIN3 is essential for the late ovule initiation. STK, the positive regulator of ovule initiation, involves in PIN3-mediated late ovule initiation. BR influences the late ovule initiation through positively regulating PIN3 expression. Taken together, we describe the intact process of ovule initiation and demonstrate the late ovule initiation contributes to the extra ovule number, which provides promising strategies for yield improvement of cruciferous and leguminous crops.

Funding: W-H. L. received the findings from the National Natural Science Foundation of China (32070342, 31771591, and 31761163003), Shanghai Jiao Tong University JiRLMDS Joint Research Fund (Project MDS-JF-2020-8), the Agri-X Interdisciplinary Fund of Shanghai Jiao Tong University (Agri-X20200204 and Agri-X2017006), the Bio-X Interdisciplinary Fund of Shanghai Jiao Tong University (20CX-04), and the Scientific and Technological Innovation Funds of Shanghai Jiao Tong University (19X160020009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Our previous work has elucidated the two groups of asynchronous ovule primordia initiation at floral stage 9 [ 11 ]. Basically, the first group of ovule primordia protrude firstly. Along with the placenta elongation, the size of interval between existing ovules enlarges, and the second group of ovule primordia initiate from these boundary regions. These two group ovules grow to similar size and shape and begin differentiating at floral stage 10. In this study, we further illustrated that most of ovules (around 90%) initiated early (in two groups) and the remaining 10% ovules protruded late. Further investigations revealed that pin3 mutants showed reduced pistil size and lacking late ovule initiation, leading to the specifically decreased extra ovule number. PIN3-mediated polar auxin transport is crucial for the late ovule initiation and STK involves in this process. In addition, BR participated in ovule initiation by positively regulating auxin signaling and PIN3 expression. Taken together, our results describe the late process of ovule initiation in A. thaliana, demonstrate the regulatory mechanism of the late ovule initiation, and provide clues for increasing ovule number and seed yield.

Considered together, the integration of multiple plant hormones regulates the ovule initiation. Furthermore, the auxin peaks directly mediate this process. Polar distribution of PIN proteins in the cell membrane enables the directional transport of intercellular auxin [ 41 ]. Among the eight members of the PIN family (PIN1–PIN8) in A. thaliana [ 42 ], PIN1 and PIN3 are mainly expressed in developing ovules [ 4 , 28 , 43 , 44 ]. Besides, PIN1 is required for ovule patterning and female gametophyte development during the later ovule developmental stages [ 33 ], while PIN3 may not play predominant role since the pin3-4 mutant shows normal ovule patterning and female gametogenesis (the publications of other alleles of pin3 mutants have not mentioned the defective ovule development) [ 43 , 45 ]. However, whether PIN3 contributes to the initiation of ovule primordia still remains unknown.

Brassinosteroid (BR) positively regulates the ovule number as well. Earlier research demonstrated that BR-deficient mutants, including cpd, bri1-116, bri1-5, bin2-1 and det2-1, have shortened pistils and produce relatively few ovules [ 38 , 39 ], whereas the enhanced BR signaling in the bzr1-1D mutant lengthens the pistil and significantly increases the ovule number by up-regulating the expression of HLL and ANT [ 39 ]. The shortened pistils of the bin2-1 mutant are partial result of the decreased auxin signaling [ 40 ], which is consistent with our report that increased BR could enhance auxin response during ovule initiation [ 11 ].

Cytokinin (CK) has also been demonstrated to regulate the ovule primordia formation. In cytokinin oxidase/dehydrogenase3 (ckx3) ckx5 double mutant, the ovule number was increased due to the raised CK content [ 34 ]. By contrast, the number of ovule primordia decreases substantially in the cytokinin response1-12(cre1-12) histidine kinase2-2 (ahk2-2) ahk3-3 and Arabidopsis thaliana response regulator1 (arr1) arr10 arr12 triple mutants, which exhibit decreased responsiveness to CK because of the altered auxin fluxes resulting from the modulated PIN1 expression during ovule development [ 33 , 35 – 37 ].

Plant hormones have important regulatory effects on the ovule number. A double mutant (yucca1 yucca4) in which auxin synthesis is adversely affected produces fewer ovules because of a compromised local auxin response [ 29 ]. A similar phenotype was also observed in the mp S319 mutant with a weakly mutated MONOPTEROS (MP), encoding a transcription factor of the auxin response factor (ARF) family [ 30 – 32 ]. Polar auxin transport is essential for the regulation of ovule initiation since the partial loss-of-function pin1-5 mutant showed dramatically reduced ovule number [ 11 , 28 , 33 ]. Moreover, MP directly targets CUC1 and CUC2 to control PIN1 expression and localization during the formation of ovule primordia [ 28 ]. Currently, we reported that the auxin flow mediated by the dynamic polar localization of auxin efflux carrier PIN1, leading to the formation of auxin maxima, is essential for asynchronous ovule initiation [ 11 ].

In A. thaliana, the ovule primordia arise asymmetrically at floral stages 8–9 by the periclinal cell divisions within the subepidermal tissue of the placenta, which develops from the two opposing meristematic ridges (also called carpel margin meristems, CMMs) and differentiates along the length of the septum adjacent to the pistil walls at floral stage 8 (floral developmental stages according to [ 1 ]) [ 2 – 13 ]. The ovule primordia protrusion can be considered as a type of lateral organ initiation [ 5 , 14 ]. To date, several key genes and genetic regulatory networks related to ovule primordia identity and initiation have been characterized. The MADS box genes APETALA2 (AP2), AGAMOUS (AG), SHATTERPROOF1/2 (SHP1/2), SEPALLATA1/2/3 (SEP1/2/3) and SEEDSTICK (STK) encode crucial regulators of ovule identity and initiation through affecting carpel development [ 15 – 19 ]. Among these genes, STK specifically expresses in placentae and ovules, and is regulated by the GA-binding protein BPC1 [ 18 , 20 , 21 ]. Additionally, mutations in other pivotal regulators, including HUELLENLOS (HLL), BELL1 (BEL1), and AINTEGUMENTA (ANT), decrease the ovule number [ 22 – 26 ]. CUC1 and CUC2 function redundantly controlling septal fusions and ovule boundaries (the interval between ovules), thereby influencing ovule identity. Moreover, cuc1 cuc2 double mutant produce fewer ovule primordia than wild-type [ 27 , 28 ].

There are many seeds in one fruit in Arabidopsis and some important crops, such as leguminous and cruciferous crops. The ovule is the precursor of the seed, and its initiation process determines the maximum ovule number per flower and has great impact on the maximum seeds per fruit, making it an important factor affecting the final seed yield of these plants.

Results

Mutations of PIN3 disrupted polar auxin transport and decreased auxin response Earlier research demonstrated that PIN3 encodes an auxin efflux carrier that mediates the polar auxin transport essential for root growth and gravitropism [45]. Therefore, the gravitropism and auxin response were investigated. The bending angle of pin3 mutants root tip was compromised upon gravi-stimulus (S2A and S2B Fig), indicating root gravitropism was impaired. Moreover, the root and hypocotyl length of pin3 mutants were substantially shorter than those of the wild-type (S2A, S2C and S2E Fig). Further investigations revealed that the defective elongation of the hypocotyl epidermal cells was responsible for the decreased hypocotyl length of the mutants (S2D–S2F Fig). These auxin-related phenotypes of pin3-1′ and pin3-2′ are in accordance with pin3-1 and pin3-2 mutants identified in previous study [45], demonstrating that pin3-1′ and pin3-2′ were knock-down mutants of PIN3. Furthermore, the reduced elongation of the wild-type hypocotyl epidermal cells induced by the application of exogenous 1-naphthylphthalamic acid (NPA), which is an inhibitor of polar auxin transport, were similar to those of pin3 mutant cells (S2D and S2F Fig), providing evidence that polar auxin efflux of pin3 mutants was disrupted. Considered together, these results verified that knocking down of PIN3 leads to the disrupted polar auxin transport and decreased auxin response.

Disruption of PIN3 leads to the compromised auxin signaling during ovule initiation DR5 is an auxin responsive element [51]. Its expression is induced by auxin and displays the cells with active auxin response [52]. DR5-NLS-eGFP has been widely used as an auxin responsive reporter [53], which provides a convenient tool to indicate the cells with auxin signaling in developing ovules [43]. To investigate whether the decreased ovule number of pin3 mutants was due to the reduced auxin signaling during ovule initiation, we separately crossed the pin3-1′ and pin3-2′ mutants with the DR5::NLS-eGFP reporter line [53]. The fluorescence observations revealed that the DR5 signal in ovule tips at floral stage 10 was obviously weaker in pin3-1′ and pin3-2′ backgrounds than that in the wild-type (Fig 4A–4F). And the analysis of the fluorescence intensity showed that DR5 signal decreased dramatically in both pin3 mutants (Fig 4G), indicating the auxin signaling decreased in the ovule tips probably because that less auxin was transported to the ovule tips in pin3 mutants. These results reflected the importance of PIN3 for the polar auxin transport required to establish auxin maximal which is sufficient for the late ovule initiation. PPT PowerPoint slide

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TIFF original image Download: Fig 4. DR5 signal is compromised in pin3 ovules at floral stage 10. (A-F) Confocal microscopy observations of the DR5::NLS-eGFP reporter in the wild-type (A-B), pin3-1’ (C-D), and pin3-2’ (E-F) backgrounds. (G) Quantification of DR5 fluorescence intensity in the ovule tip. Ten independent pistils were analysed. Bars: 50 μm. White arrows indicate the representative ovules for fluorescence intensity analysis. Data are presented as the mean ± SD. Significant differences were revealed by one-way ANOVA (*** P < 0.001). https://doi.org/10.1371/journal.pgen.1010077.g004

Application of exogenous auxin analogue affects ovule initiation and its number To demonstrate that PIN3 regulates the late ovule initiation through enhancing auxin signal, we first treated the wild-type and pin3 mutants with exogenous 1-naphthlcetic acid (NAA), an efflux substrate. This auxin analogue enters cells by passive diffusion and its accumulation level is dependent on the efflux carriers such as PIN proteins [54]. Statistical analysis showed that the ovule number per placenta of the wild-type and pin3 mutants at floral stages 9c–11 wasn’t obviously increased under NAA treatment, implying that exogenous NAA did not efficiently enhance the auxin signal that is sufficient to promote ovule initiation, regardless of the presence of a functional PIN3 in the wild-type (S3A–S3C Fig). Meanwhile, another auxin analogue, picloram was also applied to the wild-type and pin3 mutants. Picloram is transported into cells by a special native plasma membrane-bound influx carrier PIC30, a member of the major facilitator superfamily (MFS) [55,56]. Interestingly, the pistil length and ovule number of the wild-type and pin3 mutants were both significantly increased upon picloram treatment (Fig 5A–5C). Taken together, the picloram promotes ovule initiation and recovers the ovule number of pin3 mutants, providing additional evidence that the decreased ovule number in pin3 mutants may be caused by a lack of PIN3-mediated polar auxin transport. PPT PowerPoint slide

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TIFF original image Download: Fig 5. The picloram treatment recovers the ovule number of pin3 mutants by altering auxin signal. (A) DIC images of the ovule initiation at floral stage 9c. Flowers grew for 2 days following the mock solution or 5 μM picloram treatment for 24 h. (B) Pistil length at floral stage 9c under picloram treatment. (C) Ovule number per placenta at floral stage 11. (D-E) Expression pattern of DR5::NLS-eGFP under the treatments of mock solution (D) or 5 μM picloram (E). White arrows indicate the ovule tips with GFP fluorescence. (F-G) GFP fluorescence intensity in the medial domain of the pistil (F) and ovule tip (G). Bars: 20 μm (A) and 50 μm (D-E). Data are presented as the mean ± SD (n = 12). Significant differences were revealed by one-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001). https://doi.org/10.1371/journal.pgen.1010077.g005

Picloram alters the local auxin distribution in the ovule and medial region of the pistil To clarify how picloram promotes ovule initiation, we analyzed whether auxin signaling was affected upon picloram treatment. In the absence of picloram, the DR5::NLS-eGFP signal was mainly restricted to the ovule tips in agreement with the published data [11,28], indicating the maximal of auxin signal (Fig 5D). By contrast, the expression pattern of DR5::NLS-eGFP was interfered in the presence of picloram, resulting in a strong signal in the entire ovule and the medial region of the pistil (Fig 5E–5G). Therefore, the considerable auxin accumulation associated with the enhanced expression of DR5::NLS-eGFP in the medial region may explain the elongated pistil and increased ovule number in pin3 mutants upon picloram treatment. Several studies have demonstrated that PIN1 encodes a major auxin transporter that regulates ovule initiation [11,28,33,47]. Therefore, we examined whether PIN1 protein levels are affected in pin3 mutants. The western-blot result revealed that there is no significant difference of PIN1 protein levels between pin3 mutants and the wild-type (S4 Fig), indicating that the normal PIN1 protein level could not cover the phenotype of no late ovule initiation caused by knocking down of PIN3. Therefore, we deduced that PIN3 may be the main player in the late ovule initiation.

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