A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes.

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A notable example is the >150 Amaryllidaceae alkaloids (AmAs), including galantamine, an FDA-approved treatment for Alzheimer's disease. We show that while AmAs accumulate to high levels in many daffodil tissues, biosynthesis is localized to nascent, growing tissue at the leaf base. A similar trend is found in the production of steroidal alkaloids (e.g., cyclopamine) in corn lily. This model of active biosynthesis enabled the elucidation of a complete set of biosynthetic genes that can be used to produce AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodils. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes, where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as above-ground tissue develops.

Form Follows Function: How to Build a Deadly Trap.

The carnivorous plant Utricularia gibba forms cup-shaped leaflets to capture prey. Whitewoods et al. (2020) use computational modeling to simulate the formation of the trap's 3D geometry. Directional expansion of the young leaflet is proposed to be a crucial morphogenetic driver, pointing at a fundamental principle of plant development.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

Flavin Monooxygenase-Generated N-Hydroxypipecolic Acid Is a Critical Element of Plant Systemic Immunity.

Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection.

Maize smart-canopy architecture enhances yield at high densities.

Increasing planting density is a key strategy for enhancing maize yields1-3. An ideotype for dense planting requires a 'smart canopy' with leaf angles at different canopy layers differentially optimized to maximize light interception and photosynthesis4-6, among other features. Here we identified leaf angle architecture of smart canopy 1 (lac1), a natural mutant with upright upper leaves, less erect middle leaves and relatively flat lower leaves. lac1 has improved photosynthetic capacity and attenuated responses to shade under dense planting. lac1 encodes a brassinosteroid C-22 hydroxylase that predominantly regulates upper leaf angle. Phytochrome A photoreceptors accumulate in shade and interact with the transcription factor RAVL1 to promote its degradation via the 26S proteasome, thereby inhibiting activation of lac1 by RAVL1 and decreasing brassinosteroid levels. This ultimately decreases upper leaf angle in dense fields. Large-scale field trials demonstrate that lac1 boosts maize yields under high planting densities. To quickly introduce lac1 into breeding germplasm, we transformed a haploid inducer and recovered homozygous lac1 edits from 20 diverse inbred lines. The tested doubled haploids uniformly acquired smart-canopy-like plant architecture. We provide an important target and an accelerated strategy for developing high-density-tolerant cultivars, with lac1 serving as a genetic chassis for further engineering of a smart canopy in maize.

Probing plant signal processing optogenetically by two channelrhodopsins.

Early plant responses to different stress situations often encompass cytosolic Ca2+ increases, plasma membrane depolarization and the generation of reactive oxygen species1-3. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca2+ conductance that enabled triggering cytosolic Ca2+ elevations in planta. Plant responses to light-induced Ca2+ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.04. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca2+ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca2+ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

Terrestrial photosynthesis inferred from plant carbonyl sulfide uptake.

Terrestrial photosynthesis, or gross primary production (GPP), is the largest carbon flux in the biosphere, but its global magnitude and spatiotemporal dynamics remain uncertain1. The global annual mean GPP is historically thought to be around 120 PgC yr-1 (refs. 2-6), which is about 30-50 PgC yr-1 lower than GPP inferred from the oxygen-18 (18O) isotope7 and soil respiration8. This disparity is a source of uncertainty in predicting climate-carbon cycle feedbacks9,10. Here we infer GPP from carbonyl sulfide, an innovative tracer for CO2 diffusion from ambient air to leaf chloroplasts through stomata and mesophyll layers. We demonstrate that explicitly representing mesophyll diffusion is important for accurately quantifying the spatiotemporal dynamics of carbonyl sulfide uptake by plants. From the estimate of carbonyl sulfide uptake by plants, we infer a global contemporary GPP of 157 (±8.5) PgC yr-1, which is consistent with estimates from 18O (150-175 PgC yr-1) and soil respiration ( 149 - 23 + 29 PgC yr-1), but with an improved confidence level. Our global GPP is higher than satellite optical observation-driven estimates (120-140 PgC yr-1) that are used for Earth system model benchmarking. This difference predominantly occurs in the pan-tropical rainforests and is corroborated by ground measurements11, suggesting a more productive tropics than satellite-based GPP products indicated. As GPP is a primary determinant of terrestrial carbon sinks and may shape climate trajectories9,10, our findings lay a physiological foundation on which the understanding and prediction of carbon-climate feedbacks can be advanced.

Tropical forests are approaching critical temperature thresholds.

The critical temperature beyond which photosynthetic machinery in tropical trees begins to fail averages approximately 46.7 °C (Tcrit)1. However, it remains unclear whether leaf temperatures experienced by tropical vegetation approach this threshold or soon will under climate change. Here we found that pantropical canopy temperatures independently triangulated from individual leaf thermocouples, pyrgeometers and remote sensing (ECOSTRESS) have midday peak temperatures of approximately 34 °C during dry periods, with a long high-temperature tail that can exceed 40 °C. Leaf thermocouple data from multiple sites across the tropics suggest that even within pixels of moderate temperatures, upper canopy leaves exceed Tcrit 0.01% of the time. Furthermore, upper canopy leaf warming experiments (+2, 3 and 4 °C in Brazil, Puerto Rico and Australia, respectively) increased leaf temperatures non-linearly, with peak leaf temperatures exceeding Tcrit 1.3% of the time (11% for more than 43.5 °C, and 0.3% for more than 49.9 °C). Using an empirical model incorporating these dynamics (validated with warming experiment data), we found that tropical forests can withstand up to a 3.9 ± 0.5 °C increase in air temperatures before a potential tipping point in metabolic function, but remaining uncertainty in the plasticity and range of Tcrit in tropical trees and the effect of leaf death on tree death could drastically change this prediction. The 4.0 °C estimate is within the 'worst-case scenario' (representative concentration pathway (RCP) 8.5) of climate change predictions2 for tropical forests and therefore it is still within our power to decide (for example, by not taking the RCP 6.0 or 8.5 route) the fate of these critical realms of carbon, water and biodiversity3,4.

Warm springs alter timing but not total growth of temperate deciduous trees.

As the climate changes, warmer spring temperatures are causing earlier leaf-out1-3 and commencement of CO2 uptake1,3 in temperate deciduous forests, resulting in a tendency towards increased growing season length3 and annual CO2 uptake1,3-7. However, less is known about how spring temperatures affect tree stem growth8,9, which sequesters carbon in wood that has a long residence time in the ecosystem10,11. Here we show that warmer spring temperatures shifted stem diameter growth of deciduous trees earlier but had no consistent effect on peak growing season length, maximum growth rates, or annual growth, using dendrometer band measurements from 440 trees across two forests. The latter finding was confirmed on the centennial scale by 207 tree-ring chronologies from 108 forests across eastern North America, where annual ring width was far more sensitive to temperatures during the peak growing season than in the spring. These findings imply that any extra CO2 uptake in years with warmer spring temperatures4,5 does not significantly contribute to increased sequestration in long-lived woody stem biomass. Rather, contradicting projections from global carbon cycle models1,12, our empirical results imply that warming spring temperatures are unlikely to increase woody productivity enough to strengthen the long-term CO2 sink of temperate deciduous forests.

Widespread herbivory cost in tropical nitrogen-fixing tree species.

Recent observations suggest that the large carbon sink in mature and recovering forests may be strongly limited by nitrogen1-3. Nitrogen-fixing trees (fixers) in symbiosis with bacteria provide the main natural source of new nitrogen to tropical forests3,4. However, abundances of fixers are tightly constrained5-7, highlighting the fundamental unanswered question of what limits new nitrogen entering tropical ecosystems. Here we examine whether herbivory by animals is responsible for limiting symbiotic nitrogen fixation in tropical forests. We evaluate whether nitrogen-fixing trees experience more herbivory than other trees, whether herbivory carries a substantial carbon cost, and whether high herbivory is a result of herbivores targeting the nitrogen-rich leaves of fixers8,9. We analysed 1,626 leaves from 350 seedlings of 43 tropical tree species in Panama and found that: (1) although herbivory reduces the growth and survival of all seedlings, nitrogen-fixing trees undergo 26% more herbivory than non-fixers; (2) fixers have 34% higher carbon opportunity costs owing to herbivory than non-fixers, exceeding the metabolic cost of fixing nitrogen; and (3) the high herbivory of fixers is not driven by high leaf nitrogen. Our findings reveal that herbivory may be sufficient to limit tropical symbiotic nitrogen fixation and could constrain its role in alleviating nitrogen limitation on the tropical carbon sink.

Adaxial-abaxial bipolar leaf genes encode a putative cytokinin receptor and HD-Zip III, and control the formation of ectopic shoot meristems in rice.

Shoot apical meristems (SAMs) continuously initiate organ formation and maintain pluripotency through dynamic genetic regulations and cell-to-cell communications. The activity of meristems directly affects the plant's structure by determining the number and arrangement of organs and tissues. We have taken a forward genetic approach to dissect the genetic pathway that controls cell differentiation around the SAM. The rice mutants, adaxial-abaxial bipolar leaf 1 and 2 (abl1 and abl2), produce an ectopic leaf that is fused back-to-back with the fourth leaf, the first leaf produced after embryogenesis. The abaxial-abaxial fusion is associated with the formation of an ectopic shoot meristem at the adaxial base of the fourth leaf primordium. We cloned the ABL1 and ABL2 genes of rice by mapping their chromosomal positions. ABL1 encodes OsHK6, a histidine kinase, and ABL2 encodes a transcription factor, OSHB3 (Class III homeodomain leucine zipper). Expression analyses of these mutant genes as well as OSH1, a rice ortholog of the Arabidopsis STM gene, unveiled a regulatory circuit that controls the formation of an ectopic meristem near the SAM at germination.

Dual role of BdMUTE during stomatal development in the model grass Brachypodium distachyon.

Grasses form morphologically derived, four-celled stomata, where two dumbbell-shaped guard cells (GCs) are flanked by two lateral subsidiary cells (SCs). This innovative form enables rapid opening and closing kinetics and efficient plant-atmosphere gas exchange. The mobile bHLH transcription factor MUTE is required for SC formation in grasses. Yet whether and how MUTE also regulates GC development and whether MUTE mobility is required for SC recruitment is unclear. Here, we transgenically impaired BdMUTE mobility from GC to SC precursors in the emerging model grass Brachypodium distachyon. Our data indicate that reduced BdMUTE mobility severely affected the spatiotemporal coordination of GC and SC development. Furthermore, although BdMUTE has a cell-autonomous role in GC division orientation, complete dumbbell morphogenesis of GCs required SC recruitment. Finally, leaf-level gas exchange measurements showed that dosage-dependent complementation of the four-celled grass morphology was mirrored in a gradual physiological complementation of stomatal kinetics. Together, our work revealed a dual role of grass MUTE in regulating GC division orientation and SC recruitment, which in turn is required for GC morphogenesis and the rapid kinetics of grass stomata.

Single-plant-omics reveals the cascade of transcriptional changes during the vegetative-to-reproductive transition.

Plants undergo rapid developmental transitions, which occur contemporaneously with gradual changes in physiology. Moreover, individual plants within a population undergo developmental transitions asynchronously. Single-plant-omics has the potential to distinguish between transcriptional events that are associated with these binary and continuous processes. Furthermore, we can use single-plant-omics to order individual plants by their intrinsic biological age, providing a high-resolution transcriptional time series. We performed RNA-seq on leaves from a large population of wild-type Arabidopsis (Arabidopsis thaliana) during the vegetative-to-reproductive transition. Though most transcripts were differentially expressed between bolted and unbolted plants, some regulators were more closely associated with leaf size and biomass. Using a pseudotime inference algorithm, we determined that some senescence-associated processes, such as the reduction in ribosome biogenesis, were evident in the transcriptome before a bolt was visible. Even in this near-isogenic population, some variants are associated with developmental traits. These results support the use of single-plant-omics to uncover rapid transcriptional dynamics by exploiting developmental asynchrony.

Population-level exploration of alternative splicing and its unique role in controlling agronomic traits of rice.

Alternative splicing (AS) plays crucial roles in regulating various biological processes in plants. However, the genetic mechanisms underlying AS and its role in controlling important agronomic traits in rice (Oryza sativa) remain poorly understood. In this study, we explored AS in rice leaves and panicles using the rice minicore collection. Our analysis revealed a high level of transcript isoform diversity, with approximately one-fifth of the potential isoforms acting as major transcripts in both tissues. Regarding the genetic mechanism of AS, we found that the splicing of 833 genes in the leaf and 1,230 genes in the panicle was affected by cis-genetic variation. Twenty-one percent of these AS events could only be explained by large structural variations. Approximately 77.5% of genes with significant splicing quantitative trait loci (sGenes) exhibited tissue-specific regulation, and AS can cause 26.9% (leaf) and 23.6% (panicle) of sGenes to have altered, lost, or gained functional domains. Additionally, through splicing-phenotype association analysis, we identified phosphate-starvation-induced RING-type E3 ligase (OsPIE1; LOC_Os01g72480), whose splicing ratio was significantly associated with plant height. In summary, this study provides an understanding of AS in rice and its contribution to the regulation of important agronomic traits.

Genetic regulation of self-organizing azimuthal canopy orientations and their impacts on light interception in maize.

The efficiency of solar radiation interception contributes to the photosynthetic efficiency of crop plants. Light interception is a function of canopy architecture, including plant density; leaf number, length, width, and angle; and azimuthal canopy orientation. We report on the ability of some maize (Zea mays) genotypes to alter the orientations of their leaves during development in coordination with adjacent plants. Although the upper canopies of these genotypes retain the typical alternate-distichous phyllotaxy of maize, their leaves grow parallel to those of adjacent plants. A genome-wide association study (GWAS) on this parallel canopy trait identified candidate genes, many of which are associated with shade avoidance syndrome, including phytochromeC2. GWAS conducted on the fraction of photosynthetically active radiation (PAR) intercepted by canopies also identified multiple candidate genes, including liguleless1 (lg1), previously defined by its role in ligule development. Under high plant densities, mutants of shade avoidance syndrome and liguleless genes (lg1, lg2, and Lg3) exhibit altered canopy patterns, viz, the numbers of interrow leaves are greatly reduced as compared to those of nonmutant controls, resulting in dramatically decreased PAR interception. In at least the case of lg2, this phenotype is not a consequence of abnormal ligule development. Instead, liguleless gene functions are required for normal light responses, including azimuth canopy re-orientation.

GRAS transcription factor PINNATE-LIKE PENTAFOLIATA2 controls compound leaf morphogenesis in Medicago truncatula.

The milestone of compound leaf development is the generation of separate leaflet primordia during the early stages, which involves two linked but distinct morphogenetic events: leaflet initiation and boundary establishment for leaflet separation. Although some progress in understanding the regulatory pathways for each event have been made, it is unclear how they are intrinsically coordinated. Here, we identify the PINNATE-LIKE PENTAFOLIATA2 (PINNA2) gene encoding a newly identified GRAS transcription factor in Medicago truncatula. PINNA2 transcripts are preferentially detected at organ boundaries. Its loss-of-function mutations convert trifoliate leaves into a pinnate pentafoliate pattern. PINNA2 directly binds to the promoter region of the LEAFY orthologue SINGLE LEAFLET1 (SGL1), which encodes a key positive regulator of leaflet initiation, and downregulates its expression. Further analysis revealed that PINNA2 synergizes with two other repressors of SGL1 expression, the BEL1-like homeodomain protein PINNA1 and the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), to precisely define the spatiotemporal expression of SGL1 in compound leaf primordia, thereby maintaining a proper pattern of leaflet initiation. Moreover, we showed that the enriched expression of PINNA2 at the leaflet-to-leaflet boundaries is positively regulated by the boundary-specific gene MtNAM, which is essential for leaflet boundary formation. Together, these results unveil a pivotal role of the boundary-expressed transcription factor PINNA2 in regulating leaflet initiation, providing molecular insights into the coordination of intricate developmental processes underlying compound leaf pattern formation.

The GRAS transcription factor CsTL regulates tendril formation in cucumber.

Cucumber (Cucumis sativus, Cs) tendrils are slender vegetative organs that typically require manual removal to ensure orderly growth during greenhouse cultivation. Here, we identified cucumber tendril-less (tl), a Tnt1 retrotransposon-induced insertion mutant lacking tendrils. Map-based cloning identified the mutated gene, CsaV3_3G003590, which we designated as CsTL, which is homologous to Arabidopsis thaliana LATERAL SUPPRESSOR (AtLAS). Knocking out CsTL repressed tendril formation but did not affect branch initiation, whereas overexpression (OE) of CsTL resulted in the formation of two or more tendrils in one leaf axil. Although expression of two cucumber genes regulating tendril formation, Tendril (CsTEN) and Unusual Floral Organs (CsUFO), was significantly decreased in CsTL knockout lines, these two genes were not direct downstream targets of CsTL. Instead, CsTL physically interacted with CsTEN, an interaction that further enhanced CsTEN-mediated expression of CsUFO. In Arabidopsis, the CsTL homolog AtLAS acts upstream of REVOLUTA (REV) to regulate branch initiation. Knocking out cucumber CsREV inhibited branch formation without affecting tendril initiation. Furthermore, genomic regions containing CsTL and AtLAS were not syntenic between the cucumber and Arabidopsis genomes, whereas REV orthologs were found on a shared syntenic block. Our results revealed not only that cucumber CsTL possesses a divergent function in promoting tendril formation but also that CsREV retains its conserved function in shoot branching.

Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

Variation in WIDTH OF LEAF AND GRAIN contributes to grain and leaf size by controlling LARGE2 stability in rice.

Grain and flag leaf size are two important agronomic traits that influence grain yield in rice (Oryza sativa). Many quantitative trait loci (QTLs) and genes that regulate these traits individually have been identified, however, few QTLs and genes that simultaneously control these two traits have been identified. In this study, we conducted a genome-wide association analysis in rice and detected a major locus, WIDTH OF LEAF AND GRAIN (WLG), that was associated with both grain and flag leaf width. WLG encodes a RING-domain E3 ubiquitin ligase. WLGhap.B, which possesses five single nucleotide polymophysim (SNP) variations compared to WLGhap.A, encodes a protein with enhanced ubiquitination activity that confers increased rice leaf width and grain size, whereas mutation of WLG leads to narrower leaves and smaller grains. Both WLGhap.A and WLGhap.B interact with LARGE2, a HETC-type E3 ligase, however, WLGhap.B exhibits stronger interaction with LARGE2, thus higher ubiquitination activity toward LARGE2 compared with WLGhap.A. Lysine1021 is crucial for the ubiquitination of LARGE2 by WLG. Loss-of-function of LARGE2 in wlg-1 phenocopies large2-c in grain and leaf width, suggesting that WLG acts upstream of LARGE2. These findings reveal the genetic and molecular mechanism by which the WLG-LARGE2 module mediates grain and leaf size in rice and suggest the potential of WLGhap.B in improving rice yield.