Jinfang Chu

D14–SCF D3 -dependent degradation of D53 regulates strigolactone signalling

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

Insights into salt tolerance from the genome of Thellungiella salsuginea.

Thellungiella salsuginea, a close relative of Arabidopsis, represents an extremophile model for abiotic stress tolerance studies. We present the draft sequence of the T. salsuginea genome, assembled based on ∼134-fold coverage to seven chromosomes with a coding capacity of at least 28,457 genes. This genome provides resources and evidence about the nature of defense mechanisms constituting the genetic basis underlying plant abiotic stress tolerance. Comparative genomics and experimental analyses identified genes related to cation transport, abscisic acid signaling, and wax production prominent in T. salsuginea as possible contributors to its success in stressful environments.

PIF4-mediated activation of YUCCA8 expression integrates temperature into the auxin pathway in regulating Arabidopsis hypocotyl growth

Higher plants adapt their growth to high temperature by a dramatic change in plant architecture. It has been shown that the transcriptional regulator phytochrome-interacting factor 4 (PIF4) and the phytohormone auxin are involved in the regulation of high temperature–induced hypocotyl elongation in Arabidopsis. Here we report that PIF4 regulates high temperature–induced hypocotyl elongation through direct activation of the auxin biosynthetic gene YUCCA8 (YUC8). We show that high temperature co-upregulates the transcript abundance of PIF4 and YUC8. PIF4–dependency of high temperature–mediated induction of YUC8 expression as well as auxin biosynthesis, together with the finding that overexpression of PIF4 leads to increased expression of YUC8 and elevated free IAA levels in planta, suggests a possibility that PIF4 directly activates YUC8 expression. Indeed, gel shift and chromatin immunoprecipitation experiments demonstrate that PIF4 associates with the G-box–containing promoter region of YUC8. Transient expression assay in Nicotiana benthamiana leaves support that PIF4 directly activates YUC8 expression in vivo. Significantly, we show that the yuc8 mutation can largely suppress the long-hypocotyl phenotype of PIF4–overexpression plants and also can reduce high temperature–induced hypocotyl elongation. Genetic analyses reveal that the shy2-2 mutation, which harbors a stabilized mutant form of the IAA3 protein and therefore is defective in high temperature–induced hypocotyl elongation, largely suppresses the long-hypocotyl phenotype of PIF4–overexpression plants. Taken together, our results illuminate a molecular framework by which the PIF4 transcriptional regulator integrates its action into the auxin pathway through activating the expression of specific auxin biosynthetic gene. These studies advance our understanding on the molecular mechanism underlying high temperature–induced adaptation in plant architecture.

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice

Significance Premature leaf senescence is known to decrease rice yield severely, but the molecular mechanism underlying this relationship remains largely unknown. Similarly, although abscisic acid (ABA)-induced leaf senescence has long been observed, the mechanism of this pathway has yet to be determined. In this study we identified and characterized a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). The data demonstrated both that PS1/Oryza sativa NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2)-like, activated by apetala3/pistillata (OsNAP) is an ideal marker of natural senescence onset and that it functions as an important link between ABA and leaf senescence in rice. Furthermore, reduced OsNAP expression led to extended grain filling and an improved seed-setting rate, which significantly enhanced the grain yield. Thus, fine-tuning OsNAP expression should be a means of improving rice yield. It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

In this work, Kyn is identified as an auxin biosynthesis inhibitor that effectively and selectively targets TAA1-like Trp aminotransferases. Moreover, it describes a previously undiscovered positive feedback loop between auxin biosynthesis and ethylene signaling pathways in roots. The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching

Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops.

Pseudomonas syringae Effector Protein AvrB Perturbs Arabidopsis Hormone Signaling by Activating MAP Kinase 4

Pathogenic microbes often modulate phytohormone physiology in the host to their advantage. We previously showed that the Pseudomonas syringae effector protein AvrB perturbs hormone signaling, as exemplified by upregulated expression of jasmonic acid response genes, and enhances plant susceptibility. Here we show that these effects of AvrB require the Arabidopsis mitogen-activated protein kinase MAP kinase 4 (MPK4), HSP90 chaperone components, and the AvrB-interacting protein, RIN4. AvrB interacts with MPK4 and the HSP90 chaperone, and AvrB induces MPK4 activation in a manner promoted by HSP90; RIN4 likely acts downstream of MPK4. These findings link Arabidopsis proteins MPK4, HSP90, and RIN4 into a pathway that P. syringae AvrB activates for the benefit of the bacterium, perturbing hormone signaling and enhancing plant susceptibility.

Arabidopsis Tyrosylprotein Sulfotransferase Acts in the Auxin/PLETHORA Pathway in Regulating Postembryonic Maintenance of the Root Stem Cell Niche

Arabidopsis tyrosylprotein sulfotransferase (TPST) maintains the postembryonic root stem cell niche by regulating basal- and auxin-induced expression of the PLETHORA stem cell transcription factor. TPST-mediated activation of a group of sulfated peptides known as root meristem growth factors provides a link between the phytohormone auxin and PLETHORA in root stem cell niche maintenance. Recent identification of the Arabidopsis thaliana tyrosylprotein sulfotransferase (TPST) and a group of Tyr-sulfated peptides known as root meristem growth factors (RGFs) highlights the importance of protein Tyr sulfation in plant growth and development. Here, we report the action mechanism of TPST in maintenance of the root stem cell niche, which in the Arabidopsis root meristem is an area of four mitotically inactive quiescent cells plus the surrounding mitotically active stem cells. Mutation of TPST leads to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. We show that TPST expression is positively regulated by auxin and that mutation of this gene affects auxin distribution by reducing local expression levels of several PIN genes and auxin biosynthetic genes in the stem cell niche region. We also show that mutation of TPST impairs basal- and auxin-induced expression of the PLETHORA (PLT) stem cell transcription factor genes and that overexpression of PLT2 rescues the root meristem defects of the loss-of-function mutant of TPST. Together, these results support that TPST acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1 and PLT2. TPST-dependent sulfation of RGFs provides a link between auxin and PLTs in regulating root stem cell niche maintenance.

Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

Significance The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development. IAA can be synthesized through the precursor tryptophan (Trp), known as the Trp-dependent IAA biosynthetic pathway. However, IAA may also be synthesized through a proposed Trp-independent IAA biosynthetic pathway. Although the Trp-independent IAA biosynthesis was hypothesized 20 years ago, it remains a mystery. In this paper, we provide compelling evidence that the cytosol-localized indole synthase (INS) initiates the Trp-independent IAA biosynthetic pathway and that the spatial and temporal expression of INS plays an important role in the establishment of the apical–basal pattern during early embryogenesis, demonstrating that the Trp-dependent and -independent IAA biosynthetic pathways coordinately regulate embryogenesis of higher plants. The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.

CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation

A rice cytokinin oxidase/dehydrogenase gene promotes crown root formation and growth by mediating the interaction between cytokinin and auxin and is a potential target for the manipulation of rice root architecture. Crown roots constitute the majority of the rice (Oryza sativa) root system and play an important role in rice growth and development. However, the molecular mechanism of crown root formation in rice is not well understood. Here, we characterized a rice dominant mutant, root enhancer1 (ren1-D), which was observed to exhibit a more robust root system, increased crown root number, and reduced plant height. Molecular and genetic analyses revealed that these phenotypes are caused by the activation of a cytokinin oxidase/dehydrogenase (CKX) family gene, OsCKX4. Subcellular localization demonstrated that OsCKX4 is a cytosolic isoform of CKX. OsCKX4 is predominantly expressed in leaf blades and roots. It is the dominant CKX, preferentially expressed in the shoot base where crown root primordia are produced, underlining its role in root initiation. OsCKX4 is induced by exogenous auxin and cytokinin in the roots. Furthermore, one-hybrid assays revealed that OsCKX4 is a direct binding target of both the auxin response factor OsARF25 and the cytokinin response regulators OsRR2 and OsRR3. Overexpression and RNA interference of OsCKX4 confirmed that OsCKX4 plays a positive role in crown root formation. Moreover, expression analysis revealed a significant alteration in the expression of auxin-related genes in the ren1-D mutants, indicating that the OsCKX4 mediates crown root development by integrating the interaction between cytokinin and auxin. Transgenic plants harboring OsCKX4 under the control of the root-specific promoter RCc3 displayed enhanced root development without affecting their shoot parts, suggesting that this strategy could be a powerful tool in rice root engineering.