OsMKK6 Regulates Disease Resistance in Rice.

Mitogen-activated protein kinase cascades play important roles in various biological programs in plants, including immune responses, but the underlying mechanisms remain elusive. Here, we identified the lesion mimic mutant rsr25 (rust spots rice 25) and determined that the mutant harbored a loss-of-function allele for OsMKK6 (MITOGEN-ACTIVATED KINASE KINASE 6). rsr25 developed reddish-brown spots on its leaves at the heading stage, as well as on husks. Compared to the wild type, the rsr25 mutant exhibited enhanced resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae) and to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). OsMKK6 interacted with OsMPK4 (MITOGEN-ACTIVATED KINASE 4) in vivo, and OsMKK6 phosphorylated OsMPK4 in vitro. The Osmpk4 mutant is also a lesion mimic mutant, with reddish-brown spots on its leaves and husks. Pathogen-related genes were significantly upregulated in Osmpk4, and this mutant exhibited enhanced resistance to M. oryzae compared to the wild type. Our results indicate that OsMKK6 and OsMPK4 form a cascade that regulates immune responses in rice.

Transcriptome Analysis Reveals the Response Mechanism of Digitaria sanguinalis, Arabidopsis thaliana and Poa annua under 4,8-Dihydroxy-1-tetralone Treatment.

4,8-dihydroxy-l-tetralone (4,8-DHT) is an allelochemical isolated from the outer bark of Carya cathayensis that acts as a plant growth inhibitor. In order to explore the mechanism of 4,8-DHT inhibiting weed activity, we treated three species of Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua with different concentrations of 4,8-DHT and performed phenotype observation and transcriptome sequencing. The results showed that with an increase in 4,8-DHT concentration, the degree of plant damage gradually deepened. Under the same concentration of 4,8-DHT, the damage degree of leaves and roots of Digitaria sanguinalis was the greatest, followed by Arabidopsis thaliana, while Poa annua had the least damage, and the leaves turned slightly yellow. Transcriptome data showed that 24536, 9913, and 1662 differentially expressed genes (DEGs) were identified in Digitaria sanguinalis, Arabidopsis thaliana, and Poa annua, respectively. These DEGs were significantly enriched in photosynthesis, carbon fixation, glutathione metabolism, phenylpropanoid biosynthesis, and oxidative phosphorylation pathways. In addition, DEGs were also enriched in plant hormone signal transduction and the MAPK signal pathway in Arabidopsis thaliana. Further analysis showed that after 4,8-DHT treatment, the transcript levels of photosynthesis PSI- and PSII-related genes, LHCA/B-related genes, Rubisco, and PEPC were significantly decreased in Digitaria sanguinalis and Arabidopsis thaliana. At the same time, the transcription levels of genes related to glutathione metabolism and the phenylpropanoid biosynthesis pathway in Digitaria sanguinalis were also significantly decreased. However, the expression of these genes was upregulated in Arabidopsis thaliana and Poa annua. These indicated that 4,8-DHT affected the growth of the three plants through different physiological pathways, and then played a role in inhibiting plant growth. Simultaneously, the extent to which plants were affected depended on the tested plants and the content of 4,8-DHT. The identification of weed genes that respond to 4,8-DHT has helped us to further understand the inhibition of plant growth by allelochemicals and has provided a scientific basis for the development of allelochemicals as herbicides.

A novel protein domain is important for photosystem II complex assembly and photoautotrophic growth in angiosperms.

Photosystem II (PSII) is a multi-subunit protein complex of the photosynthetic electron transport chain that is vital to photosynthesis. Although the structure, composition, and function of PSII have been extensively studied, its biogenesis mechanism remains less understood. Thylakoid rhodanese-like (TROL) provides an anchor for leaf-type ferredoxin:NADP+ oxidoreductase. Here, we report the chacterizaton of a second type of TROL protein, TROL2, encoded by seed plant genomes whose function has not previously been reported. We show that TROL2 is a PSII assembly cofactor with essential roles in the establishment of photoautotrophy. TROL2 contains a 45-amino-acid domain, termed the chlorotic lethal seedling (CLS) domain, that is both necessary and sufficient for TROL2 function in PSII assembly and photoautotrophic growth. Phylogenetic analyses suggest that TROL2 may have arisen from ancestral TROL1 via gene duplication before the emergence of seed plants and acquired the CLS domain via evolution of the sequence encoding its N-terminal portion. We further reveal that TROL2 (or CLS) forms an assembly cofactor complex with the intrinsic thylakoid membrane protein LOW PSII ACCUMULATION2 and interacts with small PSII subunits to facilitate PSII complex assembly. Collectively, our study not only shows that TROL2 (CLS) is essential for photoautotrophy in angiosperms but also reveals its mechanistic role in PSII complex assembly, shedding light on the molecular and evolutionary mechanisms of photosynthetic complex assemblyin angiosperms.

Characterizing membrane anchoring of leaf-form ferredoxin-NADP+ oxidoreductase in rice.

Leaf-form ferredoxin-NADP+ oxidoreductases (LFNRs) function in the last step of the photosynthetic electron transport chain, exist as soluble proteins in the chloroplast stroma and are weakly associated with thylakoids or tightly anchored to chloroplast membranes. Arabidopsis thaliana has two LFNRs, and the chloroplast proteins AtTROL and AtTIC62 participate in anchoring AtLFNRs to the thylakoid membrane. By contrast, the membrane anchoring mechanism of rice (Oryza sativa) LFNRs has not been elucidated. Here, we investigated the membrane-anchoring mechanism of LFNRs and its physiological roles in rice. We characterized the rice protein OsTROL1 based on its homology to AtTROL. We determined that OsTROL1 is also a thylakoid membrane anchor and its loss leads to a compensatory increase in OsTIC62. OsLFNR1 attachment through a membrane anchor depends on OsLFNR2, unlike the Arabidopsis counterparts. In addition, OsTIC62 was more highly expressed in the dark than under light conditions, consistent with the increased membrane binding of OsLFNR in the dark. Moreover, we observed reciprocal stabilization between OsLFNRs and their membrane anchors. In addition, unlike in Arabidopsis, the loss of LFNR membrane anchor affects photosynthesis in rice. Overall, our study sheds light on the mechanisms anchoring LFNRs to membranes in rice and highlights differences with Arabidopsis.

Ubiquitin-Specific Protease 2 (OsUBP2) Negatively Regulates Cell Death and Disease Resistance in Rice.

Lesion mimic mutants (LMMs) are great materials for studying programmed cell death and immune mechanisms in plants. Various mechanisms are involved in the phenotypes of different LMMs, but few studies have explored the mechanisms linking deubiquitination and LMMs in rice (Oryza sativa). Here, we identified a rice LMM, rust spots rice (rsr1), resulting from the mutation of a single recessive gene. This LMM has spontaneous reddish-brown spots on its leaves, and displays enhanced resistance to both fungal leaf blast (caused by Magnaporthe oryzae) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). Map-based cloning showed that the mutated gene in rsr1 encodes a Ubiquitin-Specific Protease 2 (OsUBP2). The mutation of OsUBP2 was shown to result in reactive oxygen species (ROS) accumulation, chloroplast structural defects, and programmed cell death, while the overexpression of OsUBP2 weakened rice resistance to leaf blast. OsUBP2 is therefore a negative regulator of immune processes and ROS production. OsUBP2 has deubiquitinating enzyme activity in vitro, and the enzyme active site includes a cysteine at the 234th residue. The ubiquitinated proteomics data of rsr1 and WT provide some possible target protein candidates for OsUBP2.

OsbHLH98 regulates leaf angle in rice through transcriptional repression of OsBUL1.

Leaf angle is an important agronomic trait in cereals that helps determine plant yield by affecting planting density. However, the regulation mechanism of leaf angle remained elusive. Here, we show that OsbHLH98, a rice bHLH transcription factor, negatively regulates leaf angle. osbhlh98 mutant leaves formed a larger leaf angle, whereas transgenic plants overexpressing OsbHLH98 exhibited a slight reduction in leaf angle. We determined that the changes in leaf angle resulted from increased number and size of parenchyma cells on the adaxial side of the lamina joint in osbhlh98 mutants. Experiments using reporter constructs showed that OsbHLH98 is expressed on the adaxial side of lamina joints, consistent with its proposed function in regulating leaf angle. Furthermore, we established by chromatin immunoprecipitation and CUT&RUN that OsBUL1 is a direct downstream target of OsbHLH98. Transactivation assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated that OsbHLH98 represses OsBUL1 transcription. Our results demonstrate that OsbHLH98 negatively regulates leaf angle by counteracting brassinosteroid-induced cell elongation via the repression of OsBUL1 transcription. The characterization of OsbHLH98 and its role in determining leaf angle will lay the foundation to develop the ideal plant architecture for adaptation to high planting density.