A jasmonate-mediated regulatory network modulates diurnal floret opening time in rice.

Diurnal floret opening time (DFOT) is a pivotal trait for successful fertilization and hybrid breeding in rice. However, the molecular mechanism underlying this trait is poorly understood in rice. In this study, we combined the cytological, genetic and molecular studies to demonstrate that jasmonic acid (JA) regulates DFOT in rice through modulating the turgor and osmotic pressure of the lodicules. We show that lodicules undergo dramatic morphologic changes, accompanied by changes in water and sugar contents during the process of floret opening. Consistently, a large set of genes associated with cell osmolality and cell wall remodeling exhibits distinct expression profiles at different time points in our time-course transcriptomes of lodicules. Notably, a group of JA biosynthesis and signaling genes is continuously upregulated, accompanied by a gradual increase in JA accumulation as floret opening approaching. Furthermore, we demonstrate that the JA biosynthesis gene OsAOS1 is required for endogenous JA biosynthesis in lodicules and promoting rice DFOT. Moreover, OsMYC2, a master regulator of JA signaling, regulates rice DFOT by directly activating OsAOS1, OsSWEET4, OsPIP2;2 and OsXTH9. Collectively, our findings establish a core regulatory network mediated by JA for modulating rice DFOT and provide effective gene targets for the genetic improvement of DFOT in rice.

Targeted manipulation of grain shape genes effectively improves outcrossing rate and hybrid seed production in rice.

Stigma exsertion rate (SER) of the male sterile line is a key limiting factor for hybrid seed production in rice. Although a large number of quantitative trait loci associated with SER have been reported, few genes have been molecularly cloned and functionally characterized, severely hindering the genetic improvement of SER of the male sterile line and the breeding efficiency of hybrid rice. In this study, we identified three grain shape regulatory genes, GS3, GW8 and GS9, as potential candidate genes for targeted manipulation of grain shape and SER. We show that simultaneously knocking out these three genes could effectively increase SER by increasing the ratio of spikelet length/spikelet width and length of stigma and style, without negative impacts on other agronomic traits. Cellular examination and transcriptomic analyses revealed a role of these genes in coordinated regulation of transverse and longitudinal cell division in the pistils. Moreover, we demonstrate that targeted manipulation of these grain shape genes could significantly improve the outcrossing rate in both the ZH11 (a japonica variety) and Zhu6S (an indica male sterile line) backgrounds. Our results provide new insights into the mechanisms of rice SER regulation and develop an effective strategy to improve SER and out-crossing rate in rice, thus facilitating hybrid rice production.

Local auxin biosynthesis regulates brace root angle and lodging resistance in maize.

Root lodging poses a major threat to maize production, resulting in reduced grain yield and quality, and increased harvest costs. Here, we combined expressional, genetic, and cytological studies to demonstrate a role of ZmYUC2 and ZmYUC4 in regulating gravitropic response of the brace root and lodging resistance in maize. We show that both ZmYUC2 and ZmYUC4 are preferentially expressed in root tips with partially overlapping expression patterns, and the protein products of ZmYUC2 and ZmYUC4 are localized in the cytoplasm and endoplasmic reticulum, respectively. The Zmyuc4 single mutant and Zmyuc2/4 double mutant exhibit enlarged brace root angle compared with the wild-type plants, with larger brace root angle being observed in the Zmyuc2/4 double mutant. Consistently, the brace root tips of the Zmyuc4 single mutant and Zmyuc2/4 double mutant accumulate less auxin and are defective in proper reallocation of auxin in response to gravi-stimuli. Furthermore, we show that the Zmyuc4 single mutant and the Zmyuc2/4 double mutant display obviously enhanced root lodging resistance. Our combined results demonstrate that ZmYUC2- and ZmYUC4-mediated local auxin biosynthesis is required for normal gravity response of the brace roots and provide effective targets for breeding root lodging resistant maize cultivars.

BRASSINOSTEROID-SIGNALING KINASE1-1, a positive regulator of brassinosteroid signalling, modulates plant architecture and grain size in rice.

Brassinosteroids (BRs) are a crucial class of plant hormones that regulate plant growth and development, thus affecting many important agronomic traits in crops. However, there are still significant gaps in our understanding of the BR signalling pathway in rice. In this study, we provide multiple lines of evidence to indicate that BR-SIGNALING KINASE1-1 (OsBSK1-1) likely represents a missing component in the BR signalling pathway in rice. We showed that knockout mutants of OsBSK1-1 are less sensitive to BR and exhibit a pleiotropic phenotype, including lower plant height, less tiller number and shortened grain length, whereas transgenic plants overexpressing a gain-of-function dominant mutant form of OsBSK1-1 (OsBSK1-1A295V) are hypersensitive to BR, and exhibit some enhanced BR-responsive phenotypes. We found that OsBSK1-1 physically interacts with the BR receptor BRASSINOSTEROID INSENSITIVE1 (OsBRI1), and GLYCOGEN SYNTHASE KINASE2 (OsGSK2), a downstream component crucial for BR signalling. Moreover, we showed that OsBSK1-1 can be phosphorylated by OsBRI1 and can inhibit OsGSK2-mediated phosphorylation of BRASSINOSTEROID RESISTANT1 (OsBZR1). We further demonstrated that OsBSK1-1 genetically acts downstream of OsBRI1, but upstream of OsGSK2. Together, our results suggest that OsBSK1-1 may serve as a scaffold protein directly bridging OsBRI1 and OsGSK2 to positively regulate BR signalling, thus affecting plant architecture and grain size in rice.

Leaf angle: a target of genetic improvement in cereal crops tailored for high-density planting.

High-density planting is an effective measure for increasing crop yield per unit land area. Leaf angle (LA) is a key trait of plant architecture and a target for genetic improvement of crops. Upright leaves allow better light capture in canopy under high-density planting, thus enhancing photosynthesis efficiency, ventilation and stress resistance, and ultimately higher grain yield. Here, we summarized the latest progress on the cellular and molecular mechanisms regulating LA formation in rice and maize. We suggest several standing out questions for future studies and then propose some promising strategies to manipulate LA for breeding of cereal crops tailored for high-density planting.

DWARF53 interacts with transcription factors UB2/UB3/TSH4 to regulate maize tillering and tassel branching.

Strigolactones (SLs) are a recently identified class of phytohormones that regulate diverse developmental processes in land plants. However, the signaling mechanism of SLs in maize (Zea mays) remains largely unexplored. Here, we identified the maize gene DWARF 53 (ZmD53) and demonstrated that ZmD53 interacts with the SL receptors DWARF 14A/B (ZmD14A/B) in a rac-GR24-dependent manner. Transgenic maize plants expressing a gain-of-function mutant version of Zmd53 exhibited insensitivity to exogenous rac-GR24 treatment and a highly pleiotropic phenotype, including excess tillering and reduced tassel branching, indicating that ZmD53 functions as an authentic SL signaling repressor in maize. In addition, we showed that ZmD53 interacts with two homologous maize SPL transcription factors, UB3 and TSH4, and suppresses their transcriptional activation activity on TB1 to promote tillering. We also showed that UB2, UB3, and TSH4 can physically interact with each other and themselves, and that they can directly regulate the expression of TSH4, thus forming a positive feedback loop. Furthermore, we demonstrated that ZmD53 can repress the transcriptional activation activity of UB3 and TSH4 on their own promoters, thus decreasing tassel branch number. Our results reveal new insights into the integration of SL signaling and the miR156/SPL molecular module to coordinately regulate maize development.

ZmSPL10/14/26 are required for epidermal hair cell fate specification on maize leaf.

The epidermal hair and stomata are two types of specialized structures on the surface of plant leaves. On mature maize leaves, stomatal complexes and three types of hairs are distributed in a stereotyped pattern on the adaxial epidermis. However, the spatiotemporal relationship between epidermal hair and stomata development and the regulatory mechanisms governing their formation in maize remain largely unknown. Here, we report that three homologous ZmSPL transcription factors, ZmSPL10, ZmSPL14 and ZmSPL26, act in concert to promote epidermal hair fate on maize leaf. Cytological analyses revealed that Zmspl10/14/26 triple mutants are completely glabrous, but possess ectopic stomatal files. Strikingly, the precursor cells for prickle and bicellular hairs are transdifferentiated into ectopic stomatal complexes in the Zmspl10/14/26 mutants. Molecular analyses demonstrated that ZmSPL10/14/26 bind directly to the promoter of a WUSCHEL-related homeobox gene, ZmWOX3A, and upregulate its expression in the hair precursor cells. Moreover, several auxin-related genes are downregulated in the Zmspl10/14/26 triple mutants. Our results suggest that ZmSPL10/14/26 play a key role in promoting epidermal hair fate on maize leaves, possibly through regulating ZmWOX3A and auxin-related gene expression, and that the fates of epidermal hairs and stomata are switchable.

Cytological evidence of BSD2 functioning in both chloroplast division and dimorphic chloroplast formation in maize leaves.

BACKGROUND: Maize bsd2 (bundle sheath defective2) is a classical C4 mutant with defective C4 photosynthesis, accompanied with reduced accumulation of Rubisco (ribulose bisphosphate carboxylase oxygenase) and aberrant mature chloroplast morphology in the bundle sheath (BS) cells. However, as a hypothetical chloroplast chaperone, the effects of BSD2 on C4 chloroplast development have not been fully examined yet, which precludes a full appreciation of BSD2 function in C4 photosynthesis. The aims of our study are to find out the role ofBSD2 in regulating chloroplasts development in maize leaves, and to add new insights into our understanding of C4 biology. RESULTS: We found that at the chloroplast maturation stage, the thylakoid membranes of chloroplasts in the BS and mesophyll (M) cells became significantly looser, and the granaof chloroplasts in the M cells became thinner stacking in the bsd2 mutant when compared with the wildtype plant. Moreover, at the early chloroplast development stage, the number of dividing chloroplasts and the chloroplast division rate are both reduced in the bsd2 mutant, compared with wild type. Quantitative reverse transcriptase-PCR analysis revealed that the expression of both thylakoid formation-related genesand chloroplast division-related genes is significantly reduced in the bsd2 mutants. Further, we showed that BSD2 interacts physically with the large submit of Rubisco (LS) in Bimolecular Fluorescence Complementation assay. CONCLUSIONS: Our combined results suggest that BSD2 plays an essential role in regulating the division and differentiation of the dimorphic BS and M chloroplasts, and that it acts at a post-transcriptional level to regulate LS stability or assembly of Rubisco.

Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis.

Increasing planting density has been an effective means of increasing maize (Zea mays ssp. mays) yield per unit of land area over the past few decades. However, high-density planting will cause a reduction in the ratio of red to far-red incident light, which could trigger the shade avoidance syndrome and reduce yield. The molecular mechanisms regulating the shade avoidance syndrome are well established in Arabidopsis (Arabidopsis thaliana) but poorly understood in maize. Here, we conducted an initial functional characterization of the maize Phytochrome-Interacting Factor (PIF) gene family in regulating light signaling and photomorphogenesis. The maize genome contains seven distinct PIF genes, which could be grouped into three subfamilies: ZmPIF3s, ZmPIF4s, and ZmPIF5s Similar to the Arabidopsis PIFs, all ZmPIF proteins are exclusively localized to the nucleus and most of them can form nuclear bodies upon light irradiation. We show that all of the ZmPIF proteins could interact with ZmphyB. Heterologous expression of each ZmPIF member could partially or fully rescue the phenotype of the Arabidopsis pifq mutant, and some of these proteins conferred enhanced shade avoidance syndrome in Arabidopsis. Interestingly, all ZmPIF proteins expressed in Arabidopsis are much more stable than their Arabidopsis counterparts upon exposure to red light. Moreover, the Zmpif3, Zmpif4, and Zmpif5 knockout mutants generated via CRISPR/Cas9 technology all showed severely suppressed mesocotyl elongation in dark-grown seedlings and were less responsive to simulated shade treatment. Taken together, our results reveal both conserved and distinct molecular properties of ZmPIFs in regulating light signaling and photomorphogenesis in maize.

Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis 'Tomentosa'.

The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis 'Tomentosa', the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

Synergistic Interaction of Phytohormones in Determining Leaf Angle in Crops.

Leaf angle (LA), defined as the angle between the plant stem and leaf adaxial side of the blade, generally shapes the plant architecture into a loosen or dense structure, and thus influences the light interception and competition between neighboring plants in natural settings, ultimately contributing to the crop yield and productivity. It has been elucidated that brassinosteroid (BR) plays a dominant role in determining LA, and other phytohormones also positively or negatively participate in regulating LA. Accumulating evidences have revealed that these phytohormones interact with each other in modulating various biological processes. However, the comprehensive discussion of how the phytohormones and their interaction involved in shaping LA is relatively lack. Here, we intend to summarize the advances in the LA regulation mediated by the phytohormones and their crosstalk in different plant species, mainly in rice and maize, hopefully providing further insights into the genetic manipulation of LA trait in crop breeding and improvement in regarding to overcoming the challenge from the continuous demands for food under limited arable land area.

Isolation and Functional Characterization of a Phenylalanine Ammonia-Lyase Gene (SsPAL1) from Coleus (Solenostemon scutellarioides (L.) Codd).

Phenylalanine ammonia-lyase (PAL) is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L.) Codd), a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1), suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins) biosynthesis in Coleus at the molecular level.

Characterization of a novel DUF1618 gene family in rice.

Domain of unknown function (DUF) proteins represent a number of gene families that encode functionally uncharacterized proteins in eukaryotes. For example, DUF1618 family members in plants possess a 56-199-amino acid conserved domain and this family has not been described previously. Here, we report the characterization of 121 DUF1618 genes identified in the rice genome. Based on phylogenetic analysis, the rice DUF1618 family was divided into two major groups, each group consisting of two clades. Most DUF1618 genes with close phylogenetic relationships are located in gene clusters on the chromosomes, indicating that gene duplications increased the number of DUF1618 genes. A search for DUF1618 genes in genomic and/or expressed sequence tag databases for 35 other plant species showed that DUF1618 genes are only present in several monocot plants, suggesting that DUF1618 is a new gene family that originated after the dicot-monocot divergence. Based on public microarray databases, most rice DUF1618 genes are expressed at relatively low levels. Further experimental analysis showed that the transcriptional levels of some DUF1618 genes varied in different cultivars, and some responded to stress and hormone conditions, suggesting their important roles for development and fitness in rice (Oryza sativa L.).

Natural variation in OsMYB8 confers diurnal floret opening time divergence between indica and japonica subspecies.

The inter-subspecific indica-japonica hybrid rice confer potential higher yield than the widely used indica-indica intra-subspecific hybrid rice. Nevertheless, the utilization of this strong heterosis is currently hindered by asynchronous diurnal floret opening time (DFOT) of indica and japonica parental lines. Here, we identify OsMYB8 as a key regulator of rice DFOT. OsMYB8 induces the transcription of JA-Ile synthetase OsJAR1, thereby regulating the expression of genes related to cell osmolality and cell wall remodeling in lodicules to promote floret opening. Natural variations of OsMYB8 promoter contribute to its differential expression, thus differential transcription of OsJAR1 and accumulation of JA-Ile in lodicules of indica and japonica subspecies. Furthermore, introgression of the indica haplotype of OsMYB8 into japonica effectively promotes DFOT in japonica. Our findings reveal an OsMYB8-OsJAR1 module that regulates differential DFOT in indica and japonica, and provide a strategy for breeding early DFOT japonica to facilitate breeding of indica-japonica hybrids.

OsBSK3 Positively Regulates Grain Length and Weight by Inhibiting the Phosphatase Activity of OsPPKL1.

Brassinosteroids (BRs) are a crucial class of plant hormones that regulate many important agronomic traits in rice (Oryza sativa L.); thus, the BR signaling pathway is a very important tool for breeders to improve the grain yield and quantity of rice. Contrary to the well-established BR signaling pathway in Arabidopsis, there are significant gaps in the rice BR signaling pathway, especially the regulation mechanism from OsBSK3 to OsPPKLs and OsGSKs. In this study, we report how OsBSK3 knockout mutants confer shorter and lighter grains and exhibit a typical BR-insensitive phenotype, suggesting OsBSK3 plays a positive role in BR signaling without genetic redundancy with homologs. Furthermore, OsBSK3 could physically interact with OsPPKL1 and OsGSK3, the downstream components in BR signaling, as a scaffold protein, and inhibit the phosphatase activity of OsPPKL1 on the dephosphorylation of OsGSK3. In addition, the genetic evidence showed OsBSK3 acts upstream of OsPPKL1 in regulating grain length and weight. Our results clarify the role of OsBSK3 and provide new insights into BR-signaling mechanisms, leading to potential new targets for the genetic improvement of rice.

Improving Plant Photosynthesis through Light-Harvesting Upconversion Nanoparticles.

Nanotechnology is considered as an emerging effective means to augment plant photosynthesis. However, there is still a lot of work to be done in this field. Here, we applied the upconversion nanoparticles (UCNPs) on lettuce leaves and found that the UCNPs were able to transport into the lettuce body and colocalize with the chloroplasts. It was proved that UCNPs could harvest the near-infrared light of sunlight and increase the electron transfer rate in the photosynthesis process, thus increasing the photosynthesis rate. The gene expression analysis showed that more than 90% of gene expression in photosynthesis was upregulated. After spraying the UCNP solution on the leaves of lettuce and placing the lettuce under sunlight for 1 week, the wet/dry weight of the leaves increased by 53.33% and 45.71%, respectively. This nanoengineering of light-harvesting UCNPs may have great potential for applications in agriculture.

Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice.

Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (<2.5 cycle quantification) across tissues and developmental stages, and little fluctuation in expression in heatmaps from RiceXPro data were considered stable NRGs. To validate this strategy, we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs, and compared these genes with five established reference genes (ERGs). Only one of the ERGs (UBQ5) was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs. Therefore, public transcriptomic databases are useful for identifying NRGs. We selected two NRGs, UFC1 (Homolog of UFM1-Conjugating Enzyme 1) and FhaB (Homolog of Adhesin FhaB) for qRT-PCR analysis in rice; their homologs might be suitable for other monocot plants.

FHY3 and FAR1 Integrate Light Signals with the miR156-SPL Module-Mediated Aging Pathway to Regulate Arabidopsis Flowering.

In response to competition for light from their neighbors, shade-intolerant plants flower precociously to ensure reproductive success and survival. However, the molecular mechanisms underlying this key developmental switch are not well understood. Here, we show that a pair of Arabidopsis transcription factors essential for phytochrome A signaling, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), regulate flowering time by integrating environmental light signals with the miR156-SPL module-mediated aging pathway. We found that FHY3 and FAR1 directly interact with three flowering-promoting SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, SPL3, SPL4, and SPL5, and inhibit their binding to the promoters of several key flowering regulatory genes, including FRUITFUL (FUL), LEAFY (LFY), APETALA1 (AP1), and MIR172C, thus downregulating their transcript levels and delaying flowering. Under simulated shade conditions, levels of SPL3/4/5 proteins increase, whereas levels of FHY3 and FAR1 proteins decline, thus releasing SPL3/4/5 from FHY3/FAR1 inhibition to allow activation of FUL, LFY, AP1, and MIR172C and, consequently, early flowering. Taken together, these results unravel a novel mechanism whereby plants regulate flowering time by integrating environmental cues (such as light conditions) and an internal developmental program (the miR156-SPL module-mediated aging pathway).

Genome-wide selection and genetic improvement during modern maize breeding.

Since the development of single-hybrid maize breeding programs in the first half of the twentieth century1, maize yields have increased over sevenfold, and much of that increase can be attributed to tolerance of increased planting density2-4. To explore the genomic basis underlying the dramatic yield increase in maize, we conducted a comprehensive analysis of the genomic and phenotypic changes associated with modern maize breeding through chronological sampling of 350 elite inbred lines representing multiple eras of germplasm from both China and the United States. We document several convergent phenotypic changes in both countries. Using genome-wide association and selection scan methods, we identify 160 loci underlying adaptive agronomic phenotypes and more than 1,800 genomic regions representing the targets of selection during modern breeding. This work demonstrates the use of the breeding-era approach for identifying breeding signatures and lays the foundation for future genomics-enabled maize breeding.

Molecular mechanisms of hybrid sterility in rice.

Hybrid sterility presents a major bottleneck in hybrid crop breeding and causes postzygotic reproductive isolation in speciation. Here, we summarize the current understanding of the genetics of rice hybrid sterility and highlight new advances in deciphering the molecular basis of the major genetic loci for hybrid sterility in rice. We also discuss practical strategies for overcoming reproductive barriers to utilize hybrid vigor in inter-specific and inter-subspecific hybrid rice breeding.