Mian Gu

Balancing growth and stress responses is critical for improving crop stress tolerance. Inorganic phosphate (Pi) deficiency reduces agricultural yields. Plants have evolved a Pi-starvation response (PSR) network that coordinates growth and responds to fluctuating environmental Pi levels. Null mutations or whole-plant knockdown of PSR repressor genes, such as PHOSPHATE2 (OsPHO2) and OsSPX (Syg1, Pho81, XPR1) family genes, enhance Pi absorption and transfer but disrupt Pi homeostasis, inhibiting growth and reducing yields. To overcome this, we developed a CRISPR/Cas9 tissue-specific knockout (TSKO) system for efficient, vascular-specific somatic knockout of OsPHO2 in rice (Oryza sativa) cv. ‘Nipponbare’ across several generations. The plants showed moderately increased Pi concentrations, maintained Pi homeostasis in hydroponic culture, and increased effective tiller number and grain yield in a Pi-deficient paddy. Vascular-specific OsPHO2 knockout moderately increased OsPHO2-repressed, vascular-expressed, Pi-starvation-induced signaling in roots and alleviated disordered PSR in roots and leaves. Vascular-specific knockout of OsPHO2 or OsSPX1/2 in the Zhonghua 11 background gave similar results. Field trials confirmed the enhanced low-Pi tolerance of TSKO plants in a Pi-deficient paddy and these plants showed normal growth in a Pi-sufficient paddy. This highlights the utility of improving rice low-Pi tolerance via a tissue-specific CRISPR/Cas9 knockout, provides insights into the role of vascular tissues in PSR, and offers a promising spatial-targeting strategy for crop improvement.

The Group-I alkali metals potassium (K) and cesium (Cs) serve as an essential nutrient and a harmful element, respectively. In rice (Oryza sativa), the high-affinity K+ (HAK) transporter OsHAK1 contributes to K+ and Cs+ acquisition; however, its regulatory mechanism remains unclear. Here, we report 2 OsHAK1 regulators belonging to the APETALA2/ethylene responsive factor family, designated POTASSIUM TRANSPORTER REGULATOR 1/2 (KTR1/2). Low K+ suppresses KTR1 but induces KTR2 expression. KTR1 inhibits and KTR2 enhances OsHAK1 expression. KTR1-knockout increases and KTR2-knockout decreases K+ and Cs+ uptake. Notably, KTR2 activates KTR1 expression, and the single mutation of KTR2 has a similar effect as the double mutation of KTR1 and KTR2 in impairing rice growth and grain yield. Furthermore, OsHAK1 inactivation in ktr1 mutants or KTR2-overexpression lines reduces Cs+ content to a similar level as in the oshak1 mutant. Enhancing KTR2 expression driven by its native promoter increases grain yield and K+ uptake. Variation in K+ content in grain is associated with differences of K+ content in soils where rice accessions show a distinct combination of KTR1, KTR2, and OsHAK1 haplotypes. These results demonstrate that KTR1 and KTR2 play critical roles in the K+-mediated regulation of OsHAK1 expression, thus controlling K+ and Cs+ uptake and allowing rice to adapt to varying K+ supplies.

The precise regulation of flowering time, known as heading date in rice (Oryza sativa L.), is critical for regional adaptation, agricultural productivity, and crop rotation practices. In rice, the florigen activation complex and its downstream effectors are well-characterized mediators of the floral transition in the shoot apical meristem (SAM). Here, we characterized OsMYB110 as a SAM-localized transcription factor that promotes flowering, exhibiting functional similarity to the established flowering regulator Nhd1 (N-mediated heading date-1). Through integrated molecular and genetic analyses, we demonstrate that: (i) Nhd1 directly binds to the OsMYB110 promoter to activate its expression, while OsMYB110 in turn binds to and activates the OsMADS15 promoter to control flowering progression, and (ii) genetic epistasis places OsMYB110 downstream of Nhd1 but upstream of OsMADS15 in the flowering regulation hierarchy. Furthermore, while elevated phosphate accelerates flowering, this response is abolished in myb110 and mads15 mutants but maintained in nhd1 mutants. These results define a previously unrecognized Nhd1–OsMYB110–OsMADS15 regulatory module that integrates developmental and nutrient signaling pathways to control rice flowering time.

Upland rice is one of the most important ecotypes growing on dry or rain-fed land and exhibiting better nitrate utilization. However, the differences in nitrogen uptake and the underlying molecular mechanisms between upland and lowland rice remain unknown. In this study, a nitrate transporter gene, OsNPF7.1 , was identified by analyzing nitrogen-related gene differences in the genomic and transcriptomic profiles of the two ecotypes within the japonica subspecies. OsNPF7.1 is mainly expressed in the endodermis, xylem parenchyma cells and phloem parenchyma cells and is directly activated by OsNAC42. Ectopic expression of OsNPF7.1 in Xenopus oocytes showed that the gene functions as both a low- and high-affinity NO ₃ ^- transporter. Nitrate uptake and redistribution were impaired in npf7.1 mutants. Grain yields and nitrogen use efficiency (NUE) were elevated in OsNPF7.1 overexpression transgenic plants grown under both low and normal nitrogen supply conditions. Moreover, variation in the putative promoter increases OsNPF7.1 expression and affects nitrate acquisition and redistribution in rice. The potential elite haplotype of OsNPF7.1 in upland rice could be beneficial for breeding rice varieties with high NUE.

The high-yielding Green Revolution varieties of cereal crops are characterized by a semi-dwarf architecture and lodging resistance. Plant height is tightly regulated by the availability of phosphate (Pi), yet the underlying mechanism remains obscure. Here, we report that rice (Oryza sativa) R2R3-type MYB transcription factor MYB110 is a Pi-dependent negative regulator of plant height. MYB110 is a direct target of PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) and regulates OsPHR2-mediated inhibition of rice height. Inactivation of MYB110 increased culm diameter and bending resistance, leading to enhanced lodging resistance despite increased plant height. Strikingly, the grain yield of myb110 mutants was elevated under both high- and low-Pi regimes. Two divergent haplotypes based on single nucleotide polymorphisms in the putative promoter of MYB110 corresponded with its transcript levels and plant height in response to Pi availability. Thus, fine-tuning MYB110 expression may be a potent strategy for further increasing the yield of Green Revolution cereal crop varieties.

High-affinity potassium (K⁺) transporter (HAK)/K⁺ uptake permease (KUP)/K⁺ transporter (KT) have been identified in all genome-sequenced terrestrial plants. They play an important role in K⁺ acquisition and translocation and in enhancing salt tolerance. Here, we report that plasma-membrane-located OsHAK18 functions in K⁺ and sodium (Na⁺) circulation and sugar translocation in rice (Oryza sativa). OsHAK18 was expressed mainly, though not exclusively, in vascular tissues and particularly in the phloem. Knockout (KO) of OsHAK18 reduced K⁺ concentration in phloem sap and roots but increased K⁺ accumulation in the shoot of both Nipponbare and Zhonghua11 cultivars, while overexpression (OX) of OsHAK18 driven by its endogenous promoter increased K⁺ concentration in phloem sap and roots and promoted Na⁺ retrieval from shoot to root under salt stress. Split-root experimental analysis of rubidium (Rb⁺) uptake and circulation indicated that OsHAK18-OX promoted Rb⁺ translocation from the shoot to the root. In addition, OsHAK18-KO increased while OsHAK18-OX reduced soluble sugar content in the shoot and oppositely affected the sugar concentration in the phloem and its content in the root. Moreover, OsHAK18-OX dramatically increased grain yield and physiological K⁺ utilization efficiency. Our results suggest that – unlike other OsHAKs analyzed heretofore – OsHAK18 is critical for K⁺ and Na⁺ re-circulation from shoot to root and enhances the source-to-sink translocation of photoassimilates.

High-affinity potassium (K ⁺ ) transporters HAK/KUP/KT have been identified in all genome-sequenced terrestrial plants. They play important role in K ⁺ acquisition, translocation and enhancing salt tolerance. Here we report that the plasma-membrane-located OsHAK18 functions in K ⁺ and sodium (Na ⁺ ) circulation and sugar translocation in rice. OsHAK18 is expressed mainly, though not exclusively, in vascular tissues and particularly in the phloem. Knockout (KO) of OsHAK18 reduced K ⁺ concentration in phloem sap and in the root but increased K ⁺ accumulation in the shoot of both Nipponbare and Zhonghua11 cultivars, while overexpression (OX) of OsHAK18 driven by its endogenous promoter increased K ⁺ concentration in phloem sap and in roots, and promoted Na ⁺ retrieval from shoot to root under salt stress. Split-root experimental analysis of rubidium (Rb ⁺ ) uptake and circulation indicated that OsHAK18 -OX promoted Rb ⁺ translocation from shoot to root. In addition, OsHAK18 -KO increased while OsHAK18 -OX reduced soluble sugar content in the shoot and affected oppositely the sugar concentration in the phloem and its content in the root. Moreover, OsHAK18 -OX increased dramatically grain yield and physiological K ⁺ utilization efficiency. Taken together, our results suggest that – unlike other OsHAKs analyzed heretofore – OsHAK18 is critical to K ⁺ and Na ⁺ re-circulation from shoot to root and enhances the source-to-sink translocation of a photo-assimilate. One sentence summary: Rice potassium transporter OsHAK18 mediates shoot-to-root circulation of potassium and sodium and source-to-sink translocation of soluble sugar which improves potassium use efficiency and grain yield.

Interdependent metabolic and transport processes of carbon (C) and nitrogen (N) regulate plant growth and development, while the regulatory pathways remain poorly defined. We previously reported that a rice circadian clock N-mediated heading date-1 (Nhd1) regulates growth-duration-dependent N use efficiency. Here, we report that knockout of Nhd1 in rice reduced photosynthesis rate and sucrose ratio of sheaths to blades but increased total C to N ratio and free amino acids. Leaf RNA-seq analysis indicated that mutation of Nhd1 dramatically altered expression of the genes linking to starch and sucrose metabolism, circadian rhythm, and amino acid metabolic pathways. We identified that Nhd1 can directly activate the transcriptional expression of sucrose transporter-1 (OsSUT1). Knockout of Nhd1 suppressed OsSUT1 expression and both nhd1 and ossut1 mutants showed similar shorter height, lower shoot biomass and sucrose concentration in comparison to wild type, while overexpression of OsSUT1 can restore the defective sucrose transport and partially ameliorate the reduced growth of nhd1 mutants. Nhd1-binding site of OsSUT1 promoter is conserved in all known rice genomes. The positively related variation of Nhd1 and OsSUT1 expression among randomly selected Indica and Japonica varieties suggests a common regulatory module of Nhd1-OsSUT1 mediated C and N balance in rice.

Plants have evolved delicate systems for stimulating or inhibiting inorganic phosphate (Pi) uptake in response to the fluctuating Pi availability in soil. However, the negative regulators inhibiting Pi uptake at the transcriptional level are largely unexplored. Here, we functionally characterized a transcription factor in rice (Oryza sativa), OsWRKY10. OsWRKY10 encodes a nucleus-localized protein and showed preferential tissue localization. Knockout of OsWRKY10 led to increased Pi uptake and accumulation under Pi-replete conditions. In accordance with this phenotype, OsWRKY10 was transcriptionally induced by Pi, and a subset of PHOSPHATE TRANSPORTER 1 (PHT1) genes were up-regulated upon its mutation, suggesting that OsWRKY10 is a transcriptional repressor of Pi uptake. Moreover, rice plants expressing the OsWRKY10–VP16 fusion protein (a dominant transcriptional activator) accumulated even more Pi than oswrky10. Several lines of biochemical evidence demonstrated that OsWRKY10 directly suppressed OsPHT1;2 expression. Genetic analysis showed that OsPHT1;2 was responsible for the increased Pi accumulation in oswrky10. Furthermore, during Pi starvation, OsWRKY10 protein was degraded through the 26S proteasome. Altogether, the OsWRKY10–OsPHT1;2 module represents a crucial loop in the Pi signaling network in rice, inhibiting Pi uptake when there is ample Pi in the environment.