MADS8 is indispensable for female reproductive development at high ambient temperatures in cereal crops.

Temperature is a major factor that regulates plant growth and phenotypic diversity. To ensure reproductive success at a range of temperatures, plants must maintain developmental stability of their sexual organs when exposed to temperature fluctuations. However, the mechanisms integrating plant floral organ development and temperature responses are largely unknown. Here, we generated barley and rice loss-of-function mutants in the SEPALLATA-like MADS-box gene MADS8. The mutants in both species form multiple carpels that lack ovules at high ambient temperatures. Tissue-specific markers revealed that HvMADS8 is required to maintain floral meristem determinacy and ovule initiation at high temperatures, and transcriptome analyses confirmed that temperature-dependent differentially expressed genes in Hvmads8 mutants predominantly associate with floral organ and meristem regulation. HvMADS8 temperature-responsive activity relies on increased binding to promoters of downstream targets, as revealed by a cleavage under targets and tagmentation (CUT&Tag) analysis. We also demonstrate that HvMADS8 directly binds to 2 orthologs of D-class floral homeotic genes to activate their expression. Overall, our findings revealed a new, conserved role for MADS8 in maintaining pistil number and ovule initiation in cereal crops, extending the known function of plant MADS-box proteins in floral organ regulation.

Identification and validation of new MADS-box homologous genes in 3010 rice pan-genome.

KEY MESSAGE: Identification and validation of ten new MADS-box homologous genes in 3010 rice pan-genome for rice breeding. The functional genome is significant for rice breeding. MADS-box genes encode transcription factors that are indispensable for rice growth and development. The reported 15,362 novel genes in the rice pan-genome (RPAN) of Asian cultivated rice accessions provided a useful gene reservoir for the identification of more MADS-box candidates to overcome the limitation for the usage of only 75 MADS-box genes identified in Nipponbare for rice breeding. Here, we report the identification and validation of ten MADS-box homologous genes in RPAN. Origin and identity analysis indicated that they are originated from different wild rice accessions and structure of motif analysis revealed high variations in their amino acid sequences. Phylogenetic results with 277 MADS-box genes in 41 species showed that all these ten MADS-box homologous genes belong to type I (SRF-like, M-type). Gene expression analysis confirmed the existence of these ten MADS-box genes in IRIS_313-10,394, all of them were expressed in flower tissues, and six of them were highly expressed during seed development. Altogether, we identified and validated experimentally, for the first time, ten novel MADS-box genes in RPAN, which provides new genetic sources for rice improvement.

The regulatory role of CARBON STARVED ANTHER-mediated photoperiod-dependent male fertility in rice.

Environmental signals, especially daylength, play important roles in determining fertility in photoperiod-sensitive genic male sterile (PGMS) lines that are critical to sustain production of high-yielding hybrid rice (Oryza sativa) varieties. However, the mechanisms by which PGMS lines perceive changes in photoperiod and transmit those signals to elicit downstream effects are not well understood. In this study, we compared the transcriptomes from the leaves and anthers of carbon starved anther (csa), a PGMS line, to wild-type (WT) tissues under different photoperiods. Components of circadian clock in the leaves, including Circadian Clock-Associated 1 and Pseudo-Response Regulator (PRR95), played vital roles in sensing the photoperiod signals. Photoperiod signals were weakly transduced to anthers, where gene expression was mainly controlled by the CSA allele. CSA played a critical role in regulating sugar metabolism and cell wall synthesis in anthers under short-day conditions, and transcription of key genes inducing csa-directed sterility was upregulated under long-day (LD) conditions though not to WT levels, revealing a mechanism to explain the partial restoration of fertility in rice under LD conditions. Eight direct targets of CSA regulation were identified, all of which were genes involved in sugar metabolism and transport (cell wall invertases, SWEETs, and monosaccharide transporters) expressed only in reproductive tissues. Several hub genes coordinating the effects of CSA regulation were identified as critical elements determining WT male fertility and further analysis of these and related genes will reveal insights into how CSA coordinates sugar metabolism, cell wall biosynthesis, and photoperiod sensing in rice anther development.

Rice SIAH E3 Ligases Interact with RMD Formin and Affect Plant Morphology.

Formins are actin-binding proteins that are key to maintaining the actin cytoskeleton in cells. However, molecular mechanisms controlling the stability of formin proteins in plants remain unknown. Here, we have identified six rice SIAH-type E3 ligases, named RIP1-6 (RMD Interacting Protein 1-6) respectively, with ubiquitination enzyme activity in vitro. All six proteins can form homo- and hetero-dimers with themselves, and hetero-dimers with type II formin RMD/OsFH5. In vivo assays showed that RIP1-6 proteins localize in the cytoplasm with a punctate distribution, and all of them interact with RMD to change its native diffuse cytoplasmic localization to match that of RIP1-6. To our surprise, degradation experiments revealed that RIP1, RIP5, and RIP6 decrease rather than increase the degradation rate of RMD. Genetic analyses revealed redundancy between these six genes; either single or double mutants did not show any obvious phenotypes. However, the sextuple rip1-6 mutant displayed dwarf height, wrinkled seeds and wider leaves that were similar to the previously reported rmd mutant, and defective microfilaments and increased flag leaf angles that were not reported in rmd mutant. Collectively, our study provides insights into the mechanisms determining formin protein stability in plants.

Carbon Starved Anther modulates sugar and ABA metabolism to protect rice seed germination and seedling fitness.

Seed germination is critical for plant survival and agricultural production, which is affected by both internal seed factors and external environmental conditions. However, the genetic basis and underlying molecular mechanisms of early seed germination in crops remain largely unclear. Here, we report that R2R3 MYB transcription factor Carbon Starved Anther (CSA) is expressed specifically in Oryza sativa embryo and aleurone in response to seed imbibition, peaking at 3-6 h and undetectable by 24-h post-imbibition. CSA seeds germinated more quickly than wild-type rice seeds and had higher levels of amylase activity, glucose, and inactive abscisic acid-glucose ester (ABA-GE), but lower levels of ABA. Through analyzing the CSA-associated transcriptome and CSA binding to downstream target genes, we identified two glycolytic genes as direct CSA targets. CSA inhibits Amylase 3A expression to limit glucose production from starch and activates Os3BGlu6 expression to promote de-conjugation of ABA-GE to ABA; these functions serve to slow germination and improve seedling resilience to abiotic stress in the first 3 weeks of growth. Therefore, this study unveils a protection mechanism conferred by CSA during early seed germination by balancing glucose and ABA metabolism to optimize seed germination and stress response fitness.

Two rice MYB transcription factors maintain male fertility in response to photoperiod by modulating sugar partitioning.

Photoperiod-dependent male fertility is a critical enabler of modern hybrid breeding. A MYB transcription factor, CSA, is a key regulator of sugar partitioning in rice anthers, disruption of which causes photoperiod-sensitive male sterility. However, little is known about the molecular mechanisms governing plant fertility in response to photoperiod. Here, we have obtained another rice photoperiod-sensitive male sterile mutant, csa2, which exhibits semi-sterility under long-day (LD) conditions, with normal fertility under short-day (SD) conditions. CSA2 specifically expressed in anthers, and here is shown to be indispensable for sugar partitioning to anthers under LD conditions. The CSA2 protein can restore the fertility of csa mutants under SD conditions when expressed in a CSA-specific pattern, indicating that the two proteins share common downstream regulatory targets. Transcriptomic analyses also reveal discrete regulatory targets in anthers. Furthermore, the regulatory role of CSA2 in sugar transport was influenced by the photoperiod conditions during floral initiation, not simply during anther development. Collectively, we propose that rice evolved at least two MYB proteins, CSA2 and CSA, that regulate sugar transport in anthers under LD and SD conditions, respectively. This finding provides insight into the molecular mechanisms that regulate male fertility in response to photoperiod.

Transcriptome Analysis Reveals Photoperiod-Associated Genes Expressed in Rice Anthers.

Environmental conditions, such as photoperiod and temperature, can affect male fertility in plants. While this feature is heavily exploited in rice to generate male-sterile lines for hybrid breeding, the underlying molecular mechanisms remain largely unknown. In this study, we use a transcriptomics approach to identify key genes and regulatory networks affecting pollen maturation in rice anthers in response to different day lengths. A total of 11,726 differentially expressed genes (DEGs) were revealed, of which 177 were differentially expressed at six time points over a 24-h period. GO enrichment analysis revealed that genes at all time points were enriched in transport, carbohydrate, and lipid metabolic processes, and signaling pathways, particularly phytohormone signaling. In addition, co-expression network analysis revealed four modules strongly correlated with photoperiod. Within these four modules, 496 hub genes were identified with a high degree of connectivity to other photoperiod-sensitive DEGs, including two previously reported photoperiod- and temperature-sensitive genes affecting male fertility, Carbon Starved Anther and UDP-glucose pyrophosphorylase, respectively. This work provides a new understanding on photoperiod-sensitive pollen development in rice, and our gene expression data will provide a new, comprehensive resource to identify new environmentally sensitive genes regulating male fertility for use in crop improvement.

The heterologous expression in Arabidopsis thaliana of sorghum transcription factor SbbHLH1 downregulates lignin synthesis.

Basic helix-loop-helix (bHLH) genes are important regulators of development in plants. SbbHLH1, a Sorghum bicolor bHLH sequence, was isolated from a suppression subtractive hybridization library constructed using 13 independent brown midrib (bmr) mutants as the tester and wild-type sorghum as the driver. The gene was upregulated in at least five of the mutants at the five- to seven-leaf stage. Using a yeast expression system, the N-terminal portion of SbbHLH1 was shown to be required for proper transactivation. Its heterologous expression in Arabidopsis thaliana markedly reduced the plant's lignin content. It downregulated the lignin synthesis genes 4CL1, HCT, COMT, PAL1, and CCR1, and upregulated the transcription factors MYB83, MYB46, and MYB63. The hypothesis is proposed that SbbHLH1 has stronger effect on the regulation of lignin synthesis than the various MYB transcription factors, with a possible feedback mechanism acting on the MYB transcriptional regulators.

Identification of differentially expressed genes in sorghum (Sorghum bicolor) brown midrib mutants.

Sorghum, a species able to produce a high yield of biomass and tolerate both drought and poor soil fertility, is considered to be a potential bioenergy crop candidate. The reduced lignin content characteristic of brown midrib (bmr) mutants improves the efficiency of bioethanol conversion from biomass. Suppression subtractive hybridization combined with cDNA microarray profiling was performed to characterize differential gene expression in a set of 13 bmr mutants, which accumulate significantly less lignin than the wild-type plant BTx623. Among the 153 differentially expressed genes identified, 43 were upregulated and 110 downregulated in the mutants. A semi-quantitative RT-PCR analysis applied to 12 of these genes largely validated the microarray analysis data. The transcript abundance of genes encoding l-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase was less in the mutants than in the wild type, consistent with the expectation that both enzymes are associated with lignin synthesis. However, the gene responsible for the lignin synthesis enzyme cinnamic acid 4-hydroxylase was upregulated in the mutants, indicating that the production of monolignol from l-phenylalanine may involve more than one pathway. The identity of the differentially expressed genes could be useful for breeding sorghum with improved efficiency of bioethanol conversion from lignocellulosic biomass.