The Ubiquitin-Binding Protein OsDSK2a Mediates Seedling Growth and Salt Responses by Regulating Gibberellin Metabolism in Rice

UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins are ubiquitin receptors and transporters in the ubiquitin-proteasome system that play key roles in plant growth and development. High salinity restricts plant growth by disrupting cellular metabolism, but whether UBL-UBA proteins are involved in this process is unclear. Here, we demonstrate that the UBL-UBA protein OsDSK2a (DOMINANT SUPPRESSOR of KAR2) mediates seedling growth and salt responses in rice ( Oryza sativa ). Through analysis of osdsk2a , a mutant with retarded seedling growth, as well as in vitro and in vivo assays, we demonstrate that OsDSK2a combines with polyubiquitin chains and interacts with the gibberellin (GA)-deactivating enzyme ELONGATED UPPERMOST INTERNODE (EUI), resulting in its degradation through the ubiquitin-proteasome system. Bioactive GA levels were reduced and plant growth was retarded in the osdsk2a mutant. By contrast, eui mutants displayed increased seedling growth and bioactive GA levels. OsDSK2a levels decreased in plants under salt stress. Moreover, EUI accumulated under salt stress more rapidly in osdsk2a than in wild-type plants. Thus, OsDSK2a and EUI play opposite roles in regulating plant growth under salt stress by affecting GA metabolism. Under salt stress, OsDSK2a levels decrease, thereby increasing EUI accumulation, which promotes GA metabolism and reduces plant growth.

The ubiquitin-proteasome system plays a pivotal role in regulating plant growth and 2 abiotic stress responses (Vierstra, 2009; Wang and Deng, 2011). Protein degradation is 3 a posttranslational process that plays key roles in various biological processes 4 (Vierstra, 2003;Dreher and Callis, 2007). The degradation of a substrate protein by 5 the ubiquitin-proteasome system involves four steps: the ubiquitination, recognition, 6 delivery, and degradation of the protein by the proteasome (Tian and Xie, 2013). 7 Emerging evidence indicates that the processes by which ubiquitinated proteins are  In the current study, we demonstrate that the UBL-UBA protein OsDSK2a (a 64 homolog of DSK2) helps restrict seedling growth in rice under salt stress by 65 modulating GA catabolism. This process is mediated by the direct interaction of  with a frame-shift and premature termination of OsDSK2a, i.e., osdsk2a-c1 and 93 osdsk2a-c2, exhibited reduced seedling growth compared to the wild type ( Figure 1D).

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In addition, all osdsk2a alleles displayed retarded growth in various developmental 95 processes from the seedling to heading stages (Supplemental Figure 3E   including panicles, different internodes, nodes, leaf blades, leaf sheaths, and roots.

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OsDSK2a expression was detected in all tissues examined but primarily in dividing 116 tissues, including the uppermost internode, node, and leaf sheath ( Figure 2D). Thus,

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OsDSK2a functions throughout plant growth and development. To gain insight into the mechanism by which OsDSK2a regulates plant growth, we 122 screened for interacting factors of OsDSK2a using a yeast two-hybrid assay with 123 full-length OsDSK2a as bait. Using this system, ten proteins were confirmed to 124 interact with OsDSK2a (Supplemental Table 1). Among these, we selected  To further confirm the interaction between OsDSK2a and EUI, we performed a  Figure 5E). Thus, OsDSK2a and OsDSK2b play unequal roles in regulating plant 149 growth.

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EUI is generally expressed at low levels in most plant tissues (Luo et al., 2006), 151 suggesting that its expression is inhibited in plants at the seedling stage to ensure 152 proper plant growth. To explore this issue, we measured EUI protein levels in the 153 leaves and sheaths of seedlings. We performed cell-free assays using protein extracts 154 from the leaf or sheath tissues of transgenic plants overexpressing HA-tagged EUI 155 (EUI-HA). EUI-HA was degraded more rapidly in sheaths than in leaves 156 (Supplemental Figure 6A and 6B). Therefore, we used sheath tissue to examine EUI 157 protein stability in a subsequent assay. EUI-HA was degraded more rapidly in sheaths 158 than in leaves, which is consistent with the higher OsDSK2a transcript levels detected 159 in sheath vs. leaf tissue ( Figure 2D).

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In the cell-free assay, EUI-HA produced three bands with higher molecular masses   We expressed EUI fused to HIS tag in E. coli and incubated the tagged protein with 172 wild-type protein extracts (DJ). A poly-ubiquitin signal was observed by immunoblot 173 analysis with anti-ubiquitin antibody, together with higher molecular mass forms of 174 EUI-HIS ( Figure 4C). The ubiquitination of EUI was further confirmed by 175 co-immunoprecipitation analysis using EUI-HA fusion protein extracted from 176 EUI-HA transgenic plants ( Figure 4D), supporting the notion that the three bands of 177 EUI-HA described above were modified by ubiquitin. Therefore, EUI protein can be 178 degraded through the ubiquitin proteasome system.

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Since OsDSK2a interacts with EUI and functions as a ubiquitin-binding receptor, 180 we reasoned that OsDSK2a might mediate the degradation of EUI. To test this 181 hypothesis, we performed cell-free assays by incubating protein extracts from 182 EUI-myc transgenic plants with extracts from wild-type DJ or osdsk2a plants. 183 Measurement of EUI-myc protein levels showed that EUI-myc was more stable in 184 osdsk2a than in DJ and that MG132 inhibited the decrease in EUI-myc protein levels     Correspondingly, the eui alleles were more sensitive to salt stress than wild-type 259 plants, and the survival frequencies of eui-1 and eui-2 seedlings after salt treatment 260 were lower than those of the corresponding wild-type seedlings ( Figure 6E-6H). 261 These results demonstrate that OsDSK2a and EUI play opposite roles in salt tolerance.

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To investigate the effects of salt on seedling growth, we measured the lengths and  To confirm the effect of GA on the salt tolerance of the osdsk2a mutant, we pretreated 294 10-d-old seedlings with GA 3 for 3 d, followed by treatment with 120 mM NaCl for 2 d.

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The survival frequencies of DJ and osdsk2a seedlings under salt stress were 20% and 296 45%, respectively, which decreased to no more than 10% in the seedlings of both lines 297 subjected to GA 3 pretreatment ( Figure 7A and 7B). These results demonstrate that 298 OsDSK2a responds to salt stress by interfering with GA metabolism.

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Since GA restricts the growth of Arabidopsis plants exposed to salinity stress, a  Figure 12B and 12C). To confirm the effects of the DELLA protein 307 SLR1 on salt-inhibited growth, we explored whether GA induces the degradation of 308 SLR1 in rice (Supplemental Figure 12D and 12E). Consistently, SLR1 protein levels  Here, we uncovered a mechanism that regulates EUI at the post-transcriptional 360 levels and its role in GA homeostasis, increasing our understanding of the modulation 361 of GA metabolism. We identified OsDSK2a as an interacting protein with EUI that 362 promotes its degradation. Given that EUI is localized to the endoplasmic reticulum in OsDSK2a is a UBL-UBA protein, which E3 ligase also participates in the 370 15 ubiquitination of EUI remains to be determined. Among the proteins that interacted 371 with OsDSK2a in a yeast two-hybrid screening was a zinc finger-and RING 372 domain-containing protein of unknown function, which might have E3 ligase activity.

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The interaction between OsDSK2a and the potential E3 ligase mediating EUI 374 degradation remains to be examined. salt stress suggests that the stability of EUI modulates the inhibitory effect of salinity 400 stress on plant growth. Therefore, we propose that OsDSK2a-promoted degradation of 401 EUI serves as a control mechanism to preserve plant growth and salt stress responses.

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In the current study, we revealed that a UBL-UBA protein (a component of the    Table 2. 454 The seeds were submerged in water at 37°C for 48 h. The germinated seeds were