Low phosphorus promotes NSP1-NSP2 heterodimerization to enhance strigolactone biosynthesis and regulate shoot and root architecture in rice.

Phosphorus is an essential macronutrient for plant development and metabolism, and plants have evolved ingenious mechanisms to overcome phosphate (Pi) starvation. However, the molecular mechanisms underlying the regulation of shoot and root architecture by low phosphorus conditions and the coordinated utilization of Pi and nitrogen remain largely unclear. Here, we show that Nodulation Signaling Pathway 1 (NSP1) and NSP2 regulate rice tiller number by promoting the biosynthesis of strigolactones (SLs), a class of phytohormones with fundamental effects on plant architecture and environmental responses. We found that NSP1 and NSP2 are induced by Oryza sativa PHOSPHATE STARVATION RESPONSE2 (OsPHR2) in response to low-Pi stress and form a complex to directly bind the promoters of SL biosynthesis genes, thus markedly increasing SL biosynthesis in rice. Interestingly, the NSP1/2-SL signaling module represses the expression of CROWN ROOTLESS 1 (CRL1), a newly identified early SL-responsive gene in roots, to restrain lateral root density under Pi deficiency. We also demonstrated that GR244DO treatment under normal conditions inhibits the expression of OsNRTs and OsAMTs to suppress nitrogen absorption but enhances the expression of OsPTs to promote Pi absorption, thus facilitating the balance between nitrogen and phosphorus uptake in rice. Importantly, we found that NSP1p:NSP1 and NSP2p:NSP2 transgenic plants show improved agronomic traits and grain yield under low- and medium-phosphorus conditions. Taken together, these results revealed a novel regulatory mechanism of SL biosynthesis and signaling in response to Pi starvation, providing genetic resources for improving plant architecture and nutrient-use efficiency in low-Pi environments.

Effect of different multichannel ventilation methods on aerobic composting and vegetable waste gas emissions.

Aerobic composting, especially semipermeable membrane-covered aerobic fermentation, is known to be an effective method for recycling and reducing vegetable waste. However, this approach has rarely been applied to the aerobic composting of vegetable waste; in addition, the product characteristics and GHG emissions of the composting process have not been studied in-depth. This study investigated the effect of using different structural ventilation systems on composting efficiency and greenhouse gas emissions in a semipermeable membrane-covered vegetable waste compost. The results for the groups (MV1, MV2, and MV3) with bottom ventilation plus multichannel ventilation and the group (BV) with single bottom ventilation were compared here. The MV2 group effectively increased the average temperature by 19.06% whilst also increasing the degradation rate of organic matter by 30.81%. Additionally, the germination index value reached more than 80%, 3 days in advance. Compared to those of the BV group, the CH4, N2O, and NH3 emissions of MV2 were reduced by 32.67%, 21.52%, and 22.57%, respectively, with the total greenhouse gas emissions decreasing by 24.17%. Overall, this study demonstrated a multichannel ventilation system as a new method for improving the composting efficiency of vegetable waste whilst reducing gas emissions.

Breeding of ZhongKeFaZaoGeng1 by molecular design.

Double-cropping early-season rice is one important part of staple crop rice. In recent years, great progress has been made in breeding the double-cropping early-season japonica rice variety, ZhongKeFaZaoGeng1 (ZKFZG1), with high yield, good quality, and high resistance. The breeding of ZKFZG1 aimed at the severe problems of low quality, low income and pre-harvest sprouting in double-cropping early-season rice production, and was achieved through molecular design by selecting three parents with different beneficial genes, KongYu131, NanFangChangLiGeng, and JiGeng88 and screening for key agronomic genes in cross-breeding. ZKFZG1 has a compact plant architecture, a plant height of ~90 cm, a number of ~120 grains per panicle, a setting rate of ~85%, a 1000-grain weight of 26 grams, a yield of 8.25 t/ha, and especially good grain quality. The successful breeding of ZKFZG1 provides a new direction for double-cropping early-season rice production.

Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice.

The sedentary lifestyle and refined food consumption significantly lead to obesity, type 2 diabetes, and related complications, which have become one of the major threats to global health. This incidence could be potentially reduced by daily foods rich in resistant starch (RS). However, it remains a challenge to breed high-RS rice varieties. Here, we reported a high-RS mutant rs4 with an RS content of ~10.8% in cooked rice. The genetic study revealed that the loss-of-function SSIIIb and SSIIIa together with a strong Wx allele in the background collaboratively contributed to the high-RS phenotype of the rs4 mutant. The increased RS contents in ssIIIa and ssIIIa ssIIIb mutants were associated with the increased amylose and lipid contents. SSIIIb and SSIIIa proteins were functionally redundant, whereas SSIIIb mainly functioned in leaves and SSIIIa largely in endosperm owing to their divergent tissue-specific expression patterns. Furthermore, we found that SSIII experienced duplication in different cereals, of which one SSIII paralog was mainly expressed in leaves and another in the endosperm. SSII but not SSIV showed a similar evolutionary pattern to SSIII. The copies of endosperm-expressed SSIII and SSII were associated with high total starch contents and low RS levels in the seeds of tested cereals, compared with low starch contents and high RS levels in tested dicots. These results provided critical genetic resources for breeding high-RS rice cultivars, and the evolutionary features of these genes may facilitate to generate high-RS varieties in different cereals.

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro.

Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes. Detecting E3 ligase activity in vitro is important for exploring the molecular mechanism of protein ubiquitination. However, in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve, especially in plants, mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs. In this study, we characterized components of the rice SCFDWARF3 (SCFD3) E3 ligase, screened the coordinated E2, and reconstituted active SCFD3 E3 ligase in vitro. We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1 (eSCR) protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53, which is the key transcriptional repressor in strigolactone signaling. Finally, we replaced D3 with other F-box proteins from rice and humans and reconstituted active eSCF E3 ligases, including eSCFGID2, eSCFFBXL18, and eSCFCDC4 E3 ligases. Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins, providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size.

Crop genetic improvement requires balancing complex tradeoffs caused by gene pleiotropy and linkage drags, as exemplified by IPA1 (Ideal Plant Architecture 1), a typical pleiotropic gene in rice that increases grains per panicle but reduces tillers. In this study, we identified a 54-base pair cis-regulatory region in IPA1 via a tiling-deletion-based CRISPR-Cas9 screen that, when deleted, resolves the tradeoff between grains per panicle and tiller number, leading to substantially enhanced grain yield per plant. Mechanistic studies revealed that the deleted fragment is a target site for the transcription factor An-1 to repress IPA1 expression in panicles and roots. Targeting gene regulatory regions should help dissect tradeoff effects and provide a rich source of targets for breeding complementary beneficial traits.

SEVEN IN ABSENTIA Ubiquitin Ligases Positively Regulate Defense Against Verticillium dahliae in Gossypium hirsutum.

Ubiquitination is a post-translational regulatory mechanism that controls a variety of biological processes in plants. The E3 ligases confer specificity by recognizing target proteins for ubiquitination. Here, we identified SEVEN IN ABSENTIA (SINA) ubiquitin ligases, which belong to the RING-type E3 ligase family, in upland cotton (Gossypium hirsutum). Twenty-four GhSINAs were characterized, and the expression levels of GhSINA7, GhSINA8, and GhSINA9 were upregulated at 24 h after inoculation with Verticillium dahliae. In vitro ubiquitination assays indicated that the three GhSINAs possessed E3 ubiquitin ligase activities. Transient expression in Nicotiana benthamiana leaves showed that they localized to the nucleus. And yeast two-hybrid (Y2H) screening revealed that they could interact with each other. The ectopic overexpression of GhSINA7, GhSINA8, and GhSINA9 independently in Arabidopsis thaliana resulted in increased tolerance to V. dahliae, while individual knockdowns of GhSINA7, GhSINA8, and GhSINA9 compromised cotton resistance to the pathogen. Thus, GhSINA7, GhSINA8, and GhSINA9 act as positive regulators of defense responses against V. dahliae in cotton plants.

A route to de novo domestication of wild allotetraploid rice.

Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.

SLR1 inhibits MOC1 degradation to coordinate tiller number and plant height in rice.

The breeding of cereals with altered gibberellin (GA) signaling propelled the 'Green Revolution' by generating semidwarf plants with increased tiller number. The mechanism by which GAs promote shoot height has been studied extensively, but it is not known what causes the inverse relationship between plant height and tiller number. Here we show that rice tiller number regulator MONOCULM 1 (MOC1) is protected from degradation by binding to the DELLA protein SLENDER RICE 1 (SLR1). GAs trigger the degradation of SLR1, leading to stem elongation and also to the degradation of MOC1, and hence a decrease in tiller number. This discovery provides a molecular explanation for the coordinated control of plant height and tiller number in rice by GAs, SLR1 and MOC1.

DWARF14, A Receptor Covalently Linked with the Active Form of Strigolactones, Undergoes Strigolactone-Dependent Degradation in Rice.

Strigolactones (SLs) are the latest confirmed phytohormones that regulate shoot branching by inhibiting bud outgrowth in higher plants. Perception of SLs depends on a novel mechanism employing an enzyme-receptor DWARF14 (D14) that hydrolyzes SLs and becomes covalently modified. This stimulates the interaction between D14 and D3, leading to the ubiquitination and degradation of the transcriptional repressor protein D53. However, the regulation of SL perception in rice remains elusive. In this study, we provide evidences that D14 is ubiquitinated after SL treatment and degraded through the 26S proteasome system. The Lys280 site of the D14 amino acid sequence was important for SL-induced D14 degradation, but did not change the subcellular localization of D14 nor disturbed the interaction between D14 and D3, nor D53 degradation. Biochemical and genetic analysis indicated that the key amino acids in the catalytic center of D14 were essential for D14 degradation. We further showed that D14 degradation is dependent on D3 and is tightly correlated with protein levels of D53. These findings revealed that D14 degradation takes place following D53 degradation and functions as an important feedback regulation mechanism of SL perception in rice.

Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.).

Superior plant architecture is a key means of enhancing yield potential in high yielding varieties. A newly identified recessive gene, named sd-c, controls plant height and tiller number. Genetic analysis of an F2 population from a cross between the semi-dwarf mutant and japonica cv. Houshengheng showed that the sd-c locus was flanked by SSR markers RM27877 and RM277 on chromosome 12. Thirty nine InDel markers were developed in the region and the sd-c gene was further mapped to a 1 cM centromeric region between InDel markers C11 and C12. These sequenced markers can be used to distinguish wild type and mutants and thus can be used in marker-assisted selection. The sd-c mutant decreases culm length by about 26% and doubles the tiller number without changing seed weight. Until now only sd-1 has been used in indica rice breeding programs. The sd-c mutant seems to have no undesirable pleiotropic effects and is therefore a potential genetic resource for breeding semi-dwarf indica rice cultivars.

A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants.

The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.

Identification of a stable quantitative trait locus for percentage grains with white chalkiness in rice (Oryza sativa).

High chalkiness is a major problem in many rice-producing areas of the world, especially in hybrid rice (Oryza sativa L.) in China. We previously showed a major quantitative trait locus for the percentage of grains with white chalkiness (QTLqPGWC-8) in the interval G1149-R727 on chromosome 8 using a chromosome segment substitution line (CSSL). Here, we selected the line-CSSL50 harboring the QTLqPGWC-8 allele from the CSSLs derived from a cross between Asominori (as a recurrent parent) and IR24 (as a donor parent), which had higher percentage chalkiness, markedly different from that of Asominori. There were also significant differences in starch granules, appearance of amylose content (AAC) and milling qualities between Asominori and CSSL50, but not in grain size or thousand grain weight (TGW). The BC(4) F(2) and BC(4) F(3) populations from a cross between CSSL50 and Asominori were used for fine mapping of qPGWC-8. We narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. QTLqPGWC-8 accounted for 50.9% of the difference in PGWC between the parents. The markers tightly linked to qPGWC-8 should facilitate cloning of the gene underlying this QTL and will be of value for marker-assisted selection in breeding rice varieties with better grain quality.

Fine mapping of stable QTLs related to eating quality in rice (Oryza sativa L.) by CSSLs harboring small target chromosomal segments.

Amylose content (AC) and viscosity profile are primary indices for evaluating eating and cooking qualities of rice grain. Using chromosome segment substitution lines (CSSLs), previous studies identified a QTL cluster of genes for rice eating and cooking quality in the interval R727-G1149 on chromosome 8. In this study we report two QTLs for viscosity parameters, respectively controlling setback viscosity (SBV) and consistency viscosity (CSV), located in the same interval using rapid viscosity analyzer (RVA) profile as an indicator of eating quality. Previously reported QTL for AC was dissected into two components with opposite genetic effects. Of four QTLs, qCSV-8 and qAC-8-2 had stable genetic effects across three and four environments, respectively. qSBV-8, qCSV-8 and qAC-8-1 partly overlapped, but were separated from qAC-8-2. Based on data from an Affymetrix rice GeneChip, two genes related to starch biosynthesis at the qAC-8-2 locus were chosen for further quantitative expression analysis. Both genes showed enhanced expression in sub-CSSLs carrying the target qAC-8-2 allele, but not in sub-CSSLs without the target qAC-8-2 allele, indicating their possible role in rice quality determination. Molecular markers closely linked to the two stable QTL provide the opportunity for marker-assisted selection (MAS) in breeding high quality rice.