Alternative splicing of REGULATOR OF LEAF INCLINATION 1 modulates phosphate starvation signaling and growth in plants.

Phosphate (Pi) limitation represents a primary constraint on crop production. To better cope with Pi deficiency stress, plants have evolved multiple adaptive mechanisms for phosphorus acquisition and utilization, including the alteration of growth and the activation of Pi starvation signaling. However, how these strategies are coordinated remains largely unknown. Here, we found that the alternative splicing (AS) of REGULATOR OF LEAF INCLINATION 1 (RLI1) in rice (Oryza sativa) produces two protein isoforms: RLI1a, containing MYB DNA binding domain and RLI1b, containing both MYB and coiled-coil (CC) domains. The absence of a CC domain in RLI1a enables it to activate broader target genes than RLI1b. RLI1a, but not RLI1b, regulates both brassinolide (BL) biosynthesis and signaling by directly activating BL-biosynthesis and signaling genes. Both RLI1a and RLI1b modulate Pi starvation signaling. RLI1 and PHOSPHATE STARVATION RESPONSE 2 function redundantly to regulate Pi starvation signaling and growth in response to Pi deficiency. Furthermore, the AS of RLI1-related genes to produce two isoforms for growth and Pi signaling is widely present in both dicots and monocots. Together, these findings indicate that the AS of RLI1 is an important and functionally conserved strategy to orchestrate Pi starvation signaling and growth to help plants adapt to Pi-limitation stress.

Mitotic gene conversion can be as important as meiotic conversion in driving genetic variability in plants and other species without early germline segregation.

In contrast to common meiotic gene conversion, mitotic gene conversion, because it is so rare, is often ignored as a process influencing allelic diversity. We show that if there is a large enough number of premeiotic cell divisions, as seen in many organisms without early germline sequestration, such as plants, this is an unsafe position. From examination of 1.1 million rice plants, we determined that the rate of mitotic gene conversion events, per mitosis, is 2 orders of magnitude lower than the meiotic rate. However, owing to the large number of mitoses between zygote and gamete and because of long mitotic tract lengths, meiotic and mitotic gene conversion can be of approximately equivalent importance in terms of numbers of markers converted from zygote to gamete. This holds even if we assume a low number of premeiotic cell divisions (approximately 40) as witnessed in Arabidopsis. A low mitotic rate associated with long tracts is also seen in yeast, suggesting generality of results. For species with many mitoses between each meiotic event, mitotic gene conversion should not be overlooked.

Engineering microalgae for water phosphorus recovery to close the phosphorus cycle.

As a finite and non-renewable resource, phosphorus (P) is essential to all life and crucial for crop growth and food production. The boosted agricultural use and associated loss of P to the aquatic environment are increasing environmental pollution, harming ecosystems, and threatening future global food security. Thus, recovering and reusing P from water bodies is urgently needed to close the P cycle. As a natural, eco-friendly, and sustainable reclamation strategy, microalgae-based biological P recovery is considered a promising solution. However, the low P-accumulation capacity and P-removal efficiency of algal bioreactors restrict its application. Herein, it is demonstrated that manipulating genes involved in cellular P accumulation and signalling could triple the Chlamydomonas P-storage capacity to ~7% of dry biomass, which is the highest P concentration in plants to date. Furthermore, the engineered algae could recover P from wastewater almost three times faster than the unengineered one, which could be directly used as a P fertilizer. Thus, engineering genes involved in cellular P accumulation and signalling in microalgae could be a promising strategy to enhance P uptake and accumulation, which have the potential to accelerate the application of algae for P recovery from the water body and closing the P cycle.

Microbe-dependent and independent nitrogen and phosphate acquisition and regulation in plants.

Nitrogen (N) and phosphorus (P) are the most important macronutrients required for plant growth and development. To cope with the limited and uneven distribution of N and P in complicated soil environments, plants have evolved intricate molecular strategies to improve nutrient acquisition that involve adaptive root development, production of root exudates, and the assistance of microbes. Recently, great advances have been made in understanding the regulation of N and P uptake and utilization and how plants balance the direct uptake of nutrients from the soil with the nutrient acquisition from beneficial microbes such as arbuscular mycorrhiza. Here, we summarize the major advances in these areas and highlight plant responses to changes in nutrient availability in the external environment through local and systemic signals.

AIM1-dependent high basal salicylic acid accumulation modulates stomatal aperture in rice.

The basal levels of salicylic acid (SA) vary dramatically among plant species. In the shoot, for example, rice contains almost 100 times higher SA levels than Arabidopsis. Despite its high basal levels, neither the biosynthetic pathway nor the biological functions of SA are well understood in rice. Combining with metabolite analysis, physiological, and genetic approaches, we found that the synthesis of basal SA in rice shoot is dependent on OsAIM1, which encodes a beta-oxidation enzyme in the phenylalanine ammonia-lyase (PAL) pathway. Compromised SA accumulation in the Osaim1 mutant led to a lower shoot temperature than wild-type plants. However, this shoot temperature defect resulted from increased transpiration due to elevated steady-state stomatal aperture in the mutant. Furthermore, the high basal SA level is required for sustained expression of OsWRKY45 to modulate the steady-state stomatal aperture and shoot temperature in rice. Taken together, these results provide the direct genetic evidence for the critical role of the PAL pathway in the biosynthesis of high basal level SA in rice, which plays an important role in the regulation of steady-state stomatal aperture to promote fitness under stress conditions.

Ancient rapid functional differentiation and fixation of the duplicated members in rice Dof genes after whole genome duplication.

Whole genome duplication (WGD) in plants is typically followed by genomic downsizing, where large portions of the new genome are lost. Whether this downsizing is accompanied by increased or decreased evolutionary rates of the remaining genes is poorly known, not least because homeolog pairings are often obscured by chromosomal rearrangement. Here, we use the newly published genome from a sedge, namely Kobresia littledalei, and CRISPR/Cas-9 editing to investigate how the Rho WGD event 70 million years ago (MYA) affected transcription factor evolutionary rates, fates, and function in rice (Oryza sativa) and sorghum (Sorghum bicolor). We focus on the 30-member DNA-binding with one zinc finger (Dof) transcription factor family in both crops due to their agronomic importance. Using the known speciation dates of rice from Kobresia (97 MYA) and sorghum (50 MYA), we find that rates of amino acid substitution in the critical Dof domain region were over twofold higher during the 20-million-year period following the WGD than before or afterward. Through comparison of synteny blocks, we report that at least 11% of Dof genes were purged from 70 to 50 MYA, while only 6% have been lost in the most recent 50-million-year interval. CRISPR/Cas9 editing revealed widespread fitness-related defects in flowering and lack of redundancy of paired members, as well as significant differences in expression between gene pairs. Together these findings demonstrate the strength of Dof genes as a model for deep evolutionary study and offer one of the most detailed portraits yet of the Rho WGD impact on a gene lineage.

An integrated strategy for target SSR genotyping with toleration of nucleotide variations in the SSRs and flanking regions.

BACKGROUND: With the broad application of high-throughput sequencing and its reduced cost, simple sequence repeat (SSR) genotyping by sequencing (SSR-GBS) has been widely used for interpreting genetic data across different fields, including population genetic diversity and structure analysis, the construction of genetic maps, and the investigation of intraspecies relationships. The development of accurate and efficient typing strategies for SSR-GBS is urgently needed and several tools have been published. However, to date, no suitable accurate genotyping method can tolerate single nucleotide variations (SNVs) in SSRs and flanking regions. These SNVs may be caused by PCR and sequencing errors or SNPs among varieties, and they directly affect sequence alignment and genotyping accuracy. RESULTS: Here, we report a new integrated strategy named the accurate microsatellite genotyping tool based on targeted sequencing (AMGT-TS) and provide a user-friendly web-based platform and command-line version of AMGT-TS. To handle SNVs in the SSRs or flanking regions, we developed a broad matching algorithm (BMA) that can quickly and accurately achieve SSR typing for ultradeep coverage and high-throughput analysis of loci with SNVs compatibility and grouping of typed reads for further in-depth information mining. To evaluate this tool, we tested 21 randomly sampled loci in eight maize varieties, accompanied by experimental validation on actual and simulated sequencing data. Our evaluation showed that, compared to other tools, AMGT-TS presented extremely accurate typing results with single base resolution for both homozygous and heterozygous samples. CONCLUSION: This integrated strategy can achieve accurate SSR genotyping based on targeted sequencing, and it can tolerate single nucleotide variations in the SSRs and flanking regions. This method can be readily applied to divergent sequencing platforms and species and has excellent application prospects in genetic and population biology research. The web-based platform and command-line version of AMGT-TS are available at https://amgt-ts.plantdna.site:8445 and https://github.com/plantdna/amgt-ts , respectively.

A spatial-temporal understanding of gene regulatory networks and NtARF-mediated regulation of potassium accumulation in tobacco.

MAIN CONCLUSION: After tobacco topping, changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the NtARF genes, thus causing changes in K+ content. Tobacco (Nicotiana tabacum) is a valuable industrial and commercial crop, and the leaf is its primary product. Topping (removing apical buds) is a common agronomic practice that significantly improves the yield of tobacco leaves. Potassium (K+) plays an important physiological role in tobacco growth and leaf traits, including combustibility, aroma, and safety in cigarette products, and its levels are significantly decreased after topping. Here, to present global spatial-temporal gene expression profiles and gene regulatory networks of the core elements of K+ uptake, leaves and roots from topped and untopped plants at short- and long-term time points after topping were sampled for transcriptome analysis. We found that the wounding response was initiated in leaves in the early stages after topping. Then, in the long term, processes related to metabolism and transcription regulation, as well as ion binding and transport, were altered. The expression profiles showed that core elements of K+ uptake and xylem loading were drastically suppressed in roots after topping. Finally, transient expression experiments confirmed that changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the tobacco auxin response factor (NtARF) genes, thus causing changes in the K+ content. These results suggest that some ARFs could be selected as targets to enhance the expressions of K+ uptake transporters, leading to increment of K+ contents and improvement of leaf quality in tobacco breeding.

Insights of intracellular/intercellular phosphate transport and signaling in unicellular green algae and multicellular land plants.

Phosphorus (P) is an essential element for plant growth and development. Vacuoles play a fundamental role in the storage and remobilization of P in plants, while our understanding of the evolutionary mechanisms of creating and reusing P stores are limited. Besides, we also know very little about the coordination of intercellular P translocation, neither the inorganic phosphate (Pi) signaling nor the Pi transport patterns. Here we summarize recent advances in understanding the core elements involved in cellular and/or subcellular P homeostasis and signaling in unicellular green algae and multicellular land plants. We also propose further work that might help to uncover the high-resolution intracellular and intercellular landscape of Pi distribution and signaling in plants.

High-resolution insight into recombination events at the SD1 locus in rice.

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non-reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi-dwarf phenotype of rice in the 'Green Revolution' of the 1960s. Here, physical separation of two defects allows recombination to generate the wild-type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F2 progeny from a cross between two semi-dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10-4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single-nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype-related functional genes, which are abundant in other plant genomes.

Genomic variations and distinct evolutionary rate of rare alleles in Arabidopsis thaliana.

BACKGROUND: The variation rate in genomic regions associated with different alleles, impacts to distinct evolutionary patterns involving rare alleles. The rare alleles bias towards genome-wide association studies (GWASs), aim to detect different variants at genomic loci associated with single-nucleotide polymorphisms (SNPs) inclined to produce different haplotypes. Here, we sequenced Arabidopsis thaliana and compared its coding and non-coding genomic regions with its closest outgroup relative, Arabidopsis lyrta, which accounted for the ancestral misinference. The use of genome-wide SNPs interpret the genetic architecture of rare alleles in Arabidopsis thaliana, elucidating a significant departure from a neutral evolutionary model and the pattern of polymorphisms around a selected locus will exclusively influence natural selection. RESULTS: We found 23.4% of the rare alleles existing randomly in the genome. Notably, in our results significant differences (P < 0.01) were estimated in the relative rates between rare versus intermediate alleles, between fixed versus non-fixed mutations, and between type I versus type II rare-mutations by using the χ (2)-test. However, the rare alleles generating negative values of Tajima's D suggest that they generated under selective sweeps. Relative to polymorphic sites including SNPs, 67.5% of the fixed mutations were attributed, indicating major contributors to speciation. Substantially, an evolution occurred in the rare allele that was 1.42-times faster than that in a major haplotype. CONCLUSION: Our results interpret that rare alleles fits a random occurrence model, indicating that rare alleles occur at any locus in a genome and in any accession in a species. Based on the higher relative rate of derived to ancient mutations and higher average D xy, we conclude that rare alleles evolve faster than the higher frequency alleles. The rapid evolution of rare alleles indicates that they must have been newly generated with fixed mutations, compared with the other alleles. Eventually, PCR and sequencing results, in the flanking regions of rare allele loci confirm that they are of short extension, indicating the absence of a genome-wide pattern for a rare haplotype. The indel-associated model for rare alleles assumes that indel-associated mutations only occur in an indel heterozygote.

Cracking the code of plant central phosphate signaling.

Phosphate (Pi) is involved in numerous metabolic processes and plays a vital role in plant growth. Green plants have evolved intricate molecular bases of Pi signaling to maintain cellular Pi homeostasis. Here, we summarize recent advances in the molecular and structural bases of central Pi signaling and discuss pending questions.

The origin and evolution of salicylic acid signaling and biosynthesis in plants.

Salicylic acid (SA) plays a pivotal role in plant response to biotic and abiotic stress. Several core SA signaling regulators and key proteins in SA biosynthesis have been well characterized. However, much remains unknown about the origin, evolution, and early diversification of core elements in plant SA signaling and biosynthesis. In this study, we identified 10 core protein families in SA signaling and biosynthesis across green plant lineages. We found that the key SA signaling receptors, the nonexpresser of pathogenesis-related (NPR) proteins, originated in the most recent common ancestor (MRCA) of land plants and formed divergent groups in the ancestor of seed plants. However, key transcription factors for SA signaling, TGACG motif-binding proteins (TGAs), originated in the MRCA of streptophytes, arguing for the stepwise evolution of core SA signaling in plants. Different from the assembly of the core SA signaling pathway in the ancestor of seed plants, SA exists extensively in green plants, including chlorophytes and streptophyte algae. However, the full isochorismate synthase (ICS)-based SA synthesis pathway was first assembled in the MRCA of land plants. We further revealed that the ancient abnormal inflorescence meristem 1 (AIM1)-based ß-oxidation pathway is crucial for the biosynthesis of SA in chlorophyte algae, and this biosynthesis pathway may have facilitated the adaptation of early-diverging green algae to the high-light-intensity environment on land. Taken together, our findings provide significant insights into the early evolution and diversification of plant SA signaling and biosynthesis pathways, highlighting a crucial role of SA in stress tolerance during plant terrestrialization.

Measurement of Total Phosphorus and Polyphosphate in Chlamydomonas reinhardtii.

Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.

Comparative transcriptome analyses under individual and combined nutrient starvations provide insights into N/P/K interactions in rice.

Crops often suffer from simultaneous limitations of multiple nutrients in soils, including nitrogen (N), phosphorus (P) and potassium (K), which are three major macronutrients essential for ensuring growth and yield. Although plant responses to individual N, P, and K deficiency have been well documented, our understanding of the responses to combined nutrient deficiencies and the crosstalk between nutrient starvation responses is still limited. Here, we compared the physiological responses in rice under seven kinds of single and multiple low nutrient stress of N, P and K, and used RNA sequencing approaches to compare their transcriptome changes. A total of 13,000 genes were found to be differentially expressed under all these single and multiple low N/P/K stresses, and 66 and 174 of them were shared by all these stresses in roots and shoots, respectively. Functional enrichment analyses of the DEGs showed that a group of biological and metabolic processes were shared by these low N/P/K stresses. Comparative analyses indicated that DEGs under multiple low nutrient stress was not the simple summation of single nutrient stress. N was found to be the predominant factor affecting the transcriptome under combined nutrient stress. N, P, or K availability exhibited massive influences on the transcriptomic responses to starvation of other nutrients. Many genes involved in nutrient transport, hormone signaling, and transcriptional regulation were commonly responsive to low N/P/K stresses. Some transcription factors were predicted to regulate the expression of genes that are commonly responsive to N, P, and K starvations. These results revealed the interactions between N, P, and K starvation responses, and will be helpful for further elucidation of the molecular mechanisms underlying nutrient interactions.

Transcriptomic analysis of rapeseed (Brassica napus. L.) seed development in Xiangride, Qinghai Plateau, reveals how its special eco-environment results in high yield in high-altitude areas.

As one of the most important oil crops, rapeseed (Brassica napus) is cultivated worldwide to produce vegetable oil, animal feed, and biodiesel. As the population grows and the need for renewable energy increases, the breeding and cultivation of high-yield rapeseed varieties have become top priorities. The formation of a high rapeseed yield is so complex because it is influenced not only by genetic mechanisms but also by many environmental conditions, such as climatic conditions and different farming practices. Interestingly, many high-yield areas are located in special eco-environments, for example, in the high-altitude Xiangride area of the Qinghai Plateau. However, the molecular mechanisms underlying the formation of high yields in such a special eco-environment area remain largely unknown. Here, we conducted field yield analysis and transcriptome analysis in the Xiangride area. Compared with the yield and environmental factors in the Xinning area (a low-yielding area), we found that the relatively longer daylight length is the key to high rapeseed yield in the Xiangride area, which leads up to a 52.1% increase in rapeseed yield, especially the increase in thousand seed weight and silique number (SN). Combined with transcriptome H-cluster analysis and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses, we can assume that the grain development of rapeseed in the Xiangride area is ahead of schedule and lasts for a long time, leading to the high-yield results in the Xiangride area, confirmed by the expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR) of yield-related genes. Our results provide valuable information for further exploring the molecular mechanism underlying high yield in special ecological environments and provide a helpful reference for studying seed development characteristics in special-producing regions for Brassica napus.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants.

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.

Pleiotropic changes revealed by in situ recovery of the semi-dwarf gene sd1 in rice.

The "Green Revolution" that dramatically reduced cultivar heights and sharply boosted rice production mid-century was achieved in large part through introgression of defective alleles of Semi-Dwarf 1 (SD1), which encodes a GA20ox oxidase involved in the final steps of the synthesis of bioactive gibberellin in rice. Here, we ask whether converting the defective sd1 version in a modern semi-dwarf cultivar back to wild-type SD1 in situ recovers ancestral plant traits, and more broadly, what it reveals about pleiotropic effects of this gene. We assess these effects of SD1 restoration in three independent recombinant lines recovered from F2 progeny of a cross between 93-11 and PA64s. We then used RNA-seq to dissect gene network changes that accompanied SD1 restoration. We report that this in situ restoration of wild-type SD1 nearly doubles plant height, increases total grain yield per panicle, and elongates the second-leaf length. Comparison of expression profiles reveals changes in key nodes of the gibberellin pathway, such as OsKO1 and OsGA2ox3, and more broadly in genes related to metabolic networks, defense response, and catabolic processes. Two JA-induced genes, RIR1b and OsPR1b, are extremely down-regulated after SD1 restoration, suggesting that SD1 restoration alters the balance between GA and JA to plant growth, at the cost of degrading the defense response. This in situ approach at the SD1 locus also provides a model example that is applicable to other systems and will further understanding of gene networks underlying high-yield traits in crops.