A chromosome-level genome assembly for the amphibious plant Rorippa aquatica reveals its allotetraploid origin and mechanisms of heterophylly upon submergence.

The ability to respond to varying environments is crucial for sessile organisms such as plants. The amphibious plant Rorippa aquatica exhibits a striking type of phenotypic plasticity known as heterophylly, a phenomenon in which leaf form is altered in response to environmental factors. However, the underlying molecular mechanisms of heterophylly are yet to be fully understood. To uncover the genetic basis and analyze the evolutionary processes driving heterophylly in R. aquatica, we assembled the chromosome-level genome of the species. Comparative chromosome painting and chromosomal genomics revealed that allopolyploidization and subsequent post-polyploid descending dysploidy occurred during the speciation of R. aquatica. Based on the obtained genomic data, the transcriptome analyses revealed that ethylene signaling plays a central role in regulating heterophylly under submerged conditions, with blue light signaling acting as an attenuator of ethylene signal. The assembled R. aquatica reference genome provides insights into the molecular mechanisms and evolution of heterophylly.

Diving into the Water: Amphibious Plants as a Model for Investigating Plant Adaptations to Aquatic Environments.

Amphibious plants can grow and survive in both aquatic and terrestrial environments. This review explores the diverse adaptations that enable them to thrive in such contrasting habitats. Plants with amphibious lifestyles possess fascinating traits, and their phenotypic plasticity plays an important role in adaptations. Heterophylly, the ability to produce different leaf forms, is one such trait, with submerged leaves generally being longer, narrower, and thinner than aerial leaves. In addition to drastic changes in leaf contours, amphibious plants display significant anatomical and physiological changes, including a reduction in stomatal number and cuticle thickness and changes in photosynthesis mode. This review summarizes and compares the regulatory mechanisms and evolutionary origins of amphibious plants based on molecular biology studies actively conducted in recent years using novel model amphibious plant species. Studying amphibious plants will enhance our understanding of plant adaptations to aquatic environments. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Rewiring of hormones and light response pathways underlies the inhibition of stomatal development in an amphibious plant Rorippa aquatica underwater.

Land plants have evolved the ability to cope with submergence. Amphibious plants are adapted to both aerial and aquatic environments through phenotypic plasticity in leaf form and function, known as heterophylly. In general, underwater leaves of amphibious plants are devoid of stomata, yet their molecular regulatory mechanisms remain elusive. Using the emerging model of the Brassicaceae amphibious species Rorippa aquatica, we lay the foundation for the molecular physiological basis of the submergence-triggered inhibition of stomatal development. A series of temperature shift experiments showed that submergence-induced inhibition of stomatal development is largely uncoupled from morphological heterophylly and likely regulated by independent pathways. Submergence-responsive transcriptome analysis revealed rapid reprogramming of gene expression, exemplified by the suppression of RaSPEECHLESS and RaMUTE within 1 h and the involvement of light and hormones in the developmental switch from terrestrial to submerged leaves. Further physiological studies place ethylene as a central regulator of the submergence-triggered inhibition of stomatal development. Surprisingly, red and blue light have opposing functions in this process: blue light promotes, whereas red light inhibits stomatal development, through influencing the ethylene pathway. Finally, jasmonic acid counteracts the inhibition of stomatal development, which can be attenuated by the red light. The actions and interactions of light and hormone pathways in regulating stomatal development in R. aquatica are different from those in the terrestrial species, Arabidopsis thaliana. Thus, our work suggests that extensive rewiring events of red light to ethylene signaling might underlie the evolutionary adaption to water environment in Brassicaceae.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Leaf Cell Morphology Alternation in Response to Environmental Signals in Rorippa aquatica.

Heterophylly, the phenomenon by which plants alter leaf forms to adapt to surrounding conditions, is apparent in amphibious plant species. In response to submergence, they emerge leaves with narrower blade areas. The pathway that receives the submergence signals and the mechanism regulating leaf form via cell proliferation and/or expansion systems have not yet been fully identified yet. Our anatomical study of Rorippa aquatica, an amphibious plant that exhibits heterophylly in response to various signals, showed that leaf thickness increased upon submergence; this was caused by the expansion of mesophyll cell size. Additionally, these submergence effects were inhibited under blue-light conditions. The ANGUSTIFOLIA3 (AN3)/GROWTH-REGULATING FACTOR (GRF) pathway regulating cell proliferation and cell expansion was downregulated in response to submergence; and the response was blocked under the blue-light conditions. These results suggest that submergence and light quality determine leaf cell morphology via the AN3/GRF pathway.

A tonoplast-localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg2+ conditions.

Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing under changing environmental conditions are still not completely understood. Through large-scale genetic screening, we isolated an Arabidopsis mutant (closed stomata2 (cst2)) that is defective in stomatal opening. We cloned the causal gene (MGR1/CST2) and functionally characterized this gene. The mutant phenotype was caused by a mutation in a gene encoding an unknown protein with similarities to the human magnesium (Mg2+ ) efflux transporter ACDP/CNNM. MGR1/CST2 was localized to the tonoplast and showed transport activity for Mg2+ . This protein was constitutively and highly expressed in guard cells. Knockout of this gene resulted in stomatal closing, decreased photosynthesis and growth retardation, especially under high Mg2+ conditions, while overexpression of this gene increased stomatal opening and tolerance to high Mg2+ concentrations. Furthermore, guard cell-specific expression of MGR1/CST2 in the mutant partially restored its stomatal opening. Our results indicate that MGR1/CST2 expression in the leaf guard cells plays an important role in maintaining cytosolic Mg2+ concentrations through sequestering Mg2+ into vacuoles, which is required for stomatal opening, especially under high Mg2+ conditions.

Molecular and Biochemical Differences in Leaf Explants and the Implication for Regeneration Ability in Rorippa aquatica (Brassicaceae).

Plants have a high regeneration capacity and some plant species can regenerate clone plants, called plantlets, from detached vegetative organs. We previously outlined the molecular mechanisms underlying plantlet regeneration from Rorippa aquatica (Brassicaceae) leaf explants. However, the fundamental difference between the plant species that can and cannot regenerate plantlets from vegetative organs remains unclear. Here, we hypothesized that the viability of leaf explants is a key factor affecting the regeneration capacity of R. aquatica. To test this hypothesis, the viability of R. aquatica and Arabidopsis thaliana leaf explants were compared, with respect to the maintenance of photosynthetic activity, senescence, and immune response. Time-course analyses of photosynthetic activity revealed that R. aquatica leaf explants can survive longer than those of A. thaliana. Endogenous abscisic acid (ABA) and jasmonic acid (JA) were found at low levels in leaf explant of R. aquatica. Time-course transcriptome analysis of R. aquatica and A. thaliana leaf explants suggested that senescence was suppressed at the transcriptional level in R. aquatica. Application of exogenous ABA reduced the efficiency of plantlet regeneration. Overall, our results propose that in nature, plant species that can regenerate in nature can survive for a long time.

Micrografting device for testing systemic signaling in Arabidopsis.

Grafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot- and root-ward translocation of nicotianamine-iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants.

Molecular Basis for Natural Vegetative Propagation via Regeneration in North American Lake Cress, Rorippa aquatica (Brassicaceae).

Some plant species have a striking capacity for regeneration in nature, including regeneration of the entire individual from explants. However, due to the lack of suitable experimental models, the regulatory mechanisms of spontaneous whole plant regeneration are mostly unknown. In this study, we established a novel model system to study these mechanisms using an amphibious plant within Brassicaceae, Rorippa aquatica, which naturally undergoes vegetative propagation via regeneration from leaf fragments. Morphological and anatomical observation showed that both de novo root and shoot organogenesis occurred from the proximal side of the cut edge transversely with leaf vascular tissue. Time-series RNA-seq analysis revealed that auxin and cytokinin responses were activated after leaf amputation and that regeneration-related genes were upregulated mainly on the proximal side of the leaf explants. Accordingly, we found that both auxin and cytokinin accumulated on the proximal side. Application of a polar auxin transport inhibitor retarded root and shoot regeneration, suggesting that the enhancement of auxin responses caused by polar auxin transport enhanced de novo organogenesis at the proximal wound site. Exogenous phytohormone and inhibitor applications further demonstrated that, in R. aquatica, both auxin and gibberellin are required for root regeneration, whereas cytokinin is important for shoot regeneration. Our results provide a molecular basis for vegetative propagation via de novo organogenesis.

Stem development through vascular tissues: EPFL-ERECTA family signaling that bounces in and out of phloem.

Plant cells communicate with each other using a variety of signaling molecules. Recent studies have revealed that various types of secreted peptides, as well as phytohormones known since long ago, mediate cell-cell communication in diverse contexts of plant life. These peptides affect cellular activities, such as proliferation and cell fate decisions, through their perception by cell surface receptors located on the plasma membrane of target cells. ERECTA (ER), an Arabidopsis thaliana receptor kinase gene, was first identified as a stem growth regulator, and since then an increasing number of studies have shown that ER is involved in a wide range of developmental and physiological processes. In particular, molecular functions of ER have been extensively studied in stomatal patterning. Furthermore, the importance of ER signaling in vascular tissues of inflorescence stems, especially in phloem cells, has recently been highlighted. In this review article, first we briefly summarize the history of ER research including studies on stomatal development, then introduce ER functions in vascular tissues, and discuss its interactions with phytohormones and other receptor kinase signaling pathways. Future questions and challenges will also be addressed.

ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl.

Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled. We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth. ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation. Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.