Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

Development and validation of KASP markers for resistance to Phytophthora capsici in Capsicum annuum L.

Resistance of Capsicum annuum to Phytophthora blight is dependent on the genetic background of the resistance source and the Phytophthora capsici isolate, which poses challenges for development of generally applicable molecular markers for marker-assisted selection. In this study, the resistance to P. capsici of C. annuum was genetically mapped to chromosome 5 within a 1.68-Mb interval by genome-wide association study analysis of 237 accessions. In this candidate region, 30 KASP markers were developed using genome resequencing data for a P. capsici-resistant line (0601 M) and a susceptible line (77,013). Seven of these KASP markers, located in the coding region of a probable leucine-rich repeats receptor-like serine/threonine-protein kinase gene (Capana05g000704), were validated in the 237 accessions, which showed an average accuracy of 82.7%. The genotyping of the seven KASP markers strongly corresponded with the phenotype of 42 individual plants in a pedigree family (PC83-163) developed from the P. capsici-resistant line CM334. This research provides a set of efficient and high-throughput KASP markers for marker-assisted selection of resistance to P. capsici in C. annuum. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01367-3.

PINOID regulates floral organ development by modulating auxin transport and interacts with MADS16 in rice.

In rice (Oryza sativa L.), floral organ development is an important trait. Although a role for PINOID in regulating floral organ development was reported recently, the underlying molecular mechanism remains unclear. Here, we isolated and characterized an abnormal floral organ mutant and mapped the causative gene through an improved MutMap method. Molecular study revealed that the observed phenotype is caused by a point mutation in OsPINOID (OsPID) gene; therefore, we named the mutation as ospid-4. Our data demonstrate that OsPID interacts with OsPIN1a and OsPIN1b to regulate polar auxin transport as shown previously. Additionally, OsPID also interacts with OsMADS16 to regulate transcription during floral organ development in rice. Together, we propose a model that OsPID regulates floral organ development by modulating auxin polar transport and interaction with OsMADS16 and/or LAX1 in rice. These results provide a novel insight into the role of OsPID in regulating floral organ development of rice, especially in stigma development, which would be useful for genetic improvement of high-yield breeding of rice.

Root exudates drive interspecific facilitation by enhancing nodulation and N2 fixation.

Plant diversity in experimental systems often enhances ecosystem productivity, but the mechanisms causing this overyielding are only partly understood. Intercropping faba beans (Vicia faba L.) and maize (Zea mays L.) result in overyielding and also, enhanced nodulation by faba beans. By using permeable and impermeable root barriers in a 2-y field experiment, we show that root-root interactions between faba bean and maize significantly increase both nodulation and symbiotic N2 fixation in intercropped faba bean. Furthermore, root exudates from maize promote faba bean nodulation, whereas root exudates from wheat and barley do not. Thus, a decline of soil nitrate concentrations caused by intercropped cereals is not the sole mechanism for maize promoting faba bean nodulation. Intercropped maize also caused a twofold increase in exudation of flavonoids (signaling compounds for rhizobia) in the systems. Roots of faba bean treated with maize root exudates exhibited an immediate 11-fold increase in the expression of chalcone-flavanone isomerase (involved in flavonoid synthesis) gene together with a significantly increased expression of genes mediating nodulation and auxin response. After 35 d, faba beans treated with maize root exudate continued to show up-regulation of key nodulation genes, such as early nodulin 93 (ENOD93), and promoted nitrogen fixation. Our results reveal a mechanism for how intercropped maize promotes nitrogen fixation of faba bean, where maize root exudates promote flavonoid synthesis in faba bean, increase nodulation, and stimulate nitrogen fixation after enhanced gene expression. These results indicate facilitative root-root interactions and provide a mechanism for a positive relationship between species diversity and ecosystem productivity.

Gnp4/LAX2, a RAWUL protein, interferes with the OsIAA3-OsARF25 interaction to regulate grain length via the auxin signaling pathway in rice.

Grain length is one of the determinants of yield in rice and auxin plays an important role in regulating it by mediating cell growth. Although several genes in the auxin pathway are involved in regulating grain length, the underlying molecular mechanisms remain unclear. In this study we identify a RING-finger and wd40-associated ubiquitin-like (RAWUL) domain-containing protein, Gnp4/LAX2, with a hitherto unknown role in regulation of grain length by its influence on cell expansion. Gnp4/LAX2 is broadly expressed in the plant and subcellular localization analysis shows that it encodes a nuclear protein. Overexpression of Gnp4/LAX2 can significantly increase grain length and thousand-kernel weight. Moreover, Gnp4/LAX2 physically interacts with OsIAA3 and consequently interferes with the OsIAA3-OsARF25 interaction in vitro and in vivo. OsIAA3 RNAi plants consistently exhibit longer grains, while the mutant osarf25 has small grains. In addition, OsARF25 binds to the promoter of OsERF142/SMOS1, a regulator of organ size, and positively regulates its expression. Taken together, the results reveal that Gnp4/LAX2 functions as a regulator of grain length through participation in the OsIAA3-OsARF25-OsERF142 pathway and that it has potential value for molecular breeding in rice.

Aberrant Meiotic Modulation Partially Contributes to the Lower Germination Rate of Pollen Grains in Maize (Zea mays L.) Under Low Nitrogen Supply.

Pollen germination is an essential step towards successful pollination during maize reproduction. How low niutrogen (N) affects pollen germination remains an interesting biological question to be addressed. We found that only low N resulted in a significantly lower germination rate of pollen grains after 4 weeks of low N, phosphorus or potassium treatment in maize production. Importantly, cytological analysis showed 7-fold more micronuclei in male meiocytes under the low N treatment than in the control, indicating that the lower germination rate of pollen grains was partially due to numerous chromosome loss events resulting from preceding meiosis. The appearance of 10 bivalents in the control and low N cells at diakinesis suggested that chromosome pairing and recombination in meiosis I was not affected by low N. Further gene expression analysis revealed dramatic down-regulation of Nuclear Division Cycle 80 (Ndc80) and Regulator of Chromosome Condensation 1 (Rcc1-1) expression and up-regulation of Cell Division Cycle 20 (Cdc20-1) expression, although no significant difference in the expression level of kinetochore foundation proteins Centromeric Histone H3 (Cenh3) and Centromere Protein C (Cenpc) and cohesion regulators Recombination 8 (Rec8) and Shugoshin (Sgo1) was observed. Aberrant modulation of three key meiotic regulators presumably resulted in a high likelihood of erroneous chromosome segregation, as testified by pronounced lagging chromosomes at anaphase I or cell cycle disruption at meiosis II. Thus, we proposed a cytogenetic mechanism whereby low N affects male meiosis and causes a higher chromosome loss frequency and eventually a lower germination rate of pollen grains in a staple crop plant.

Developing an Optimized Protocol for Regeneration and Transformation in Pepper.

Capsicum annuum L. is extensively cultivated in subtropical and temperate regions globally, respectively, when grown in a medium with 8 holding significant economic importance. Despite the availability of genome sequences and editing tools, gene editing in peppers is limited by the lack of a stable regeneration and transformation method. This study assessed regeneration and transformation protocols in seven chili pepper varieties, including CM334, Zunla-1, Zhongjiao6 (ZJ6), 0818, 0819, 297, and 348, in order to enhance genetic improvement efforts. Several explants, media compositions, and hormonal combinations were systematically evaluated to optimize the in vitro regeneration process across different chili pepper varieties. The optimal concentrations for shoot formation, shoot elongation, and rooting in regeneration experiments were determined as 5 mg/L of 6-Benzylaminopurine (BAP) with 5 mg/L of silver nitrate (AgNO3), 0.5 mg/L of Gibberellic acid (GA3), and 1 mg/L of Indole-3-butyric acid (IBA), respectively. The highest regeneration rate of 41% was observed from CM334 cotyledon explants. Transformation optimization established 300 mg/L of cefotaxime for bacterial control, with a 72-h co-cultivation period at OD600 = 0.1. This study optimizes the protocols for chili pepper regeneration and transformation, thereby contributing to genetic improvement efforts.

Fine Mapping and Candidate Gene Analysis of Two Major Quantitative Trait Loci, qFW2.1 and qFW3.1, Controlling Fruit Weight in Pepper (Capsicum annuum).

Fruit weight is an important agronomic trait in pepper production and is closely related to yield. At present, many quantitative trait loci (QTL) related to fruit weight have been found in pepper; however, the genes affecting fruit weight remain unknown. We analyzed the fruit weight-related quantitative traits in an intraspecific Capsicum annuum cross between the cultivated species blocky-type pepper, cv. Qiemen, and the bird pepper accession, "129-1" (Capsicum annuum var. glatriusculum), which was the wild progenitor of C. annuum. Using the QTL-seq combined with the linkage-based QTL mapping approach, QTL detection was performed; and two major effects of QTL related to fruit weight, qFW2.1 and qFW3.1, were identified on chromosomes 2 and 3. The qFW2.1 maximum explained 12.28% of the phenotypic variance observed in two F2 generations, with the maximum LOD value of 11.02, respectively; meanwhile, the qFW3.1 maximum explained 15.50% of the observed phenotypic variance in the two F2 generations, with the maximum LOD value of 11.36, respectively. qFW2.1 was narrowed down to the 1.22 Mb region using homozygous recombinant screening from BC2S2 and BC2S3 populations, while qFW3.1 was narrowed down to the 4.61Mb region. According to the transcriptome results, a total of 47 and 86 differentially expressed genes (DEGs) in the candidate regions of qFW2.1 and qFW3.1 were identified. Further, 19 genes were selected for a qRT-PCR analysis based on sequence difference combined with the gene annotation. Finally, Capana02g002938 and Capana02g003021 are the most likely candidate genes for qFW2.1, and Capana03g000903 may be a candidate gene for qFW3.1. Taken together, our results identified and fine-mapped two major QTL for fruit weight in pepper that will facilitate marker-assistant breeding for the manipulation of yield in pepper.

AtOPR3 specifically inhibits primary root growth in Arabidopsis under phosphate deficiency.

The primary root plays essential roles in root development, nutrient absorption, and root architectural establishment. Primary root growth is generally suppressed by phosphate (P) deficiency in A. thaliana; however, the underlying molecular mechanisms are largely elusive to date. We found that AtOPR3 specifically inhibited primary root growth under P deficiency via suppressing root tip growth at the transcriptional level, revealing an important novel function of AtOPR3 in regulating primary root response to the nutrient stress. Importantly, AtOPR3 functioned to down-regulate primary root growth under P limitation mostly by its own, rather than depending on the Jasmonic acid signaling pathway. Further, AtOPR3 interacted with ethylene and gibberellin signaling pathways to regulate primary root growth upon P deficiency. In addition, the AtOPR3's function in inhibiting primary root growth upon P limitation was also partially dependent on auxin polar transport. Together, our studies provide new insights into how AtOPR3, together with hormone signaling interactions, modulates primary root growth in coping with the environmental stress in Arabidopsis.