Experimental determination and mathematical modeling of standard shapes of forming autophagosomes.

The formation of autophagosomes involves dynamic morphological changes of a phagophore from a flat membrane cisterna into a cup-shaped intermediate and a spherical autophagosome. However, the physical mechanism behind these morphological changes remains elusive. Here, we determine the average shapes of phagophores by statistically investigating three-dimensional electron micrographs of more than 100 phagophores. The results show that the cup-shaped structures adopt a characteristic morphology; they are longitudinally elongated, and the rim is catenoidal with an outwardly recurved shape. To understand these characteristic shapes, we establish a theoretical model of the shape of entire phagophores. The model quantitatively reproduces the average morphology and reveals that the characteristic shape of phagophores is primarily determined by the relative size of the open rim to the total surface area. These results suggest that the seemingly complex morphological changes during autophagosome formation follow a stable path determined by elastic bending energy minimization.

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

De-differentiation in cultures of organoids from luminal-type breast cancer is restored by inhibition of NOTCH signaling.

Estrogen receptor (ER) expression in breast cancer can change during progression and the treatment, but the mechanism has not been well studied. In this study, we successfully prepared organoids from samples obtained from 33 luminal-type breast cancer patients and studied their ER expression. The expression status was well maintained in primary organoids, whereas it decreased after passaging in most of the cases. In fact, the studied organoid lines were classified into those that retained a high level of ER expression (9%), those that completely lost it (9%), and those that repressed it to varying degrees (82%). In some cases, the ER expression was suddenly and drastically decreased after passaging. Marker protein immunohistochemistry revealed that after passaging, the differentiation status shifted from a luminal- to a basal-like status. Differentially expressed genes suggested the activation of NOTCH signaling in the passaged organoids, wherein a NOTCH inhibitor was able to substantially rescue the decreased ER expression and alter the differentiation status. Our findings suggest that the differentiation status of luminal-type cancer cells is quite flexible, and that by inhibiting the NOTCH signaling we can preserve the differentiation status of luminal-type breast cancer organoids.

Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s.

Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of ß-propeller associated neurodegeneration.

ß-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21 and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including ß-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3 and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

Quantitative 3D correlative light and electron microscopy of organelle association during autophagy.

In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material. Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using three-dimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome-organelle relationships during macroautophagy.Key words: 3D-CLEM, autophagosome, electron microscopy, endoplasmic reticulum, lysosome.

NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and microferritinophagy.

A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).

Evaluation of artificial intelligence using time-lapse images of IVF embryos to predict live birth.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the prediction of live births based on embryo images? DESIGN: The AI system was created by using the Attention Branch Network associated with deep learning to predict the probability of live birth from 141,444 images recorded by time-lapse imaging of 470 transferred embryos, of which 91 resulted in live birth and 379 resulted in non-live birth that included implantation failure, biochemical pregnancy and clinical miscarriage. The possibility that the calculated confidence scores of each embryo and the focused areas visualized in each embryo image can help predict subsequent live birth was examined. RESULTS: The AI system for the first time successfully visualized embryo features in focused areas that had potential to distinguish between live and non-live births. No visual feature of embryos were visualized that were associated with live or non-live births, although there were many images in which high-focused areas existed around the zona pellucida. When a cut-off level for the confidence score was set at 0.341, the live birth rate was significantly greater for embryos with a score higher than the cut-off level than for those with a score lower than the cut-off level (P < 0.001). In addition, the live birth rate of embryos with good morphological quality and confidence scores higher than 0.341 was 41.1%. CONCLUSIONS: The authors have created an AI system with a confidence score that is useful for non-invasive selection of embryos that could result in live birth. Further study is necessary to improve selection accuracy.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Should I bend or should I grow: the mechanisms of droplet-mediated autophagosome formation.

Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.

Wetting regulates autophagy of phase-separated compartments and the cytosol.

Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.

A Missense Mutation in the NSF Gene Causes Abnormal Golgi Morphology in Arabidopsis thaliana.

The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope. At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.Key words: Golgi morphology, N-ethylmaleimide-sensitive factor (NSF), Arabidopsis thaliana.

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials.

Clinical utility of decorin in follicular fluid as a biomarker of oocyte potential.

This study investigated the concentration of decorin (DCN) in mature follicular fluid and the existence in the granulosa cells. It also investigated whether DCN is useful as a biomarker for outcomes of assisted reproductive technology (ART). A retrospective cohort study was performed involving 130 oocytes of 88 patients treated with ART because of unexplained infertility. The concentration of DCN in the follicular fluid (F-DCN) was 39.26ng/ml (median value); it was higher than that in serum. F-DCN of the oocytes fertilized by intracytoplasmic sperm injection (ICSI) was significantly lower than that of oocytes that were not fertilized (33.24ng/ml vs 40.18ng/ml; P=0.043). When a cut-off level of 34.5ng/ml was set according to the receiver-operating characteristic curve, the fertilization rate of the oocytes from the follicles in which F-DCN was lower than the cut-off level tended to be good compared to that of the oocytes with F-DCN higher than the cut-off level (P=0.052). DCN is less likely to be produced by the granulosa cells (GCs), because it was not detected in GCs by immunostaining and Western blot analysis. F-DCN has a possibility to be a biomarker indicating the quality of oocytes collected from the corresponding follicle.

Evolutionarily conserved BIL4 suppresses the degradation of brassinosteroid receptor BRI1 and regulates cell elongation.

Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.

Delineation of six species of the primitive algal genus Glaucocystis based on in situ ultrastructural characteristics.

The field of microbiology was established in the 17(th) century upon the discovery of microorganisms by Antonie van Leeuwenhoek using a single-lens microscope. Now, the detailed ultrastructures of microorganisms can be elucidated in situ using three-dimensional electron microscopy. Since the availability of electron microscopy, the taxonomy of microscopic organisms has entered a new era. Here, we established a new taxonomic system of the primitive algal genus Glaucocystis (Glaucophyta) using a new-generation electron microscopic methodology: ultra-high-voltage electron microscopy (UHVEM) and field-emission scanning electron microscopy (FE-SEM). Various globally distributed Glaucocystis strains were delineated into six species, based on differences in in situ ultrastructural features of the protoplast periphery under UHVEM tomography and in the mother cell wall by FE-SEM, as well as differences in the light microscopic characteristics and molecular phylogenetic results. The present work on Glaucocystis provides a model case of new-generation taxonomy.

A new type of 3-D peripheral ultrastructure in Glaucocystis (Glaucocystales, Glaucophyta) as revealed by ultra-high voltage electron microscopy.

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three-dimensional (3-D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra-high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3-D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3-D ultrastructure resolved in "G. incrassata" SAG 229-2 cells by 3-D modeling based on UHVEM tomography using high-pressure freezing and freeze-substitution fixation. The plasma membrane and underlying flattened vesicles in "G. incrassata" SAG 229-2 exhibited grooves at intervals of 200-600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3-D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3-D ultrastructure of the protoplast periphery is discussed.

Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells.

Phosphate Treatment Strongly Inhibits New Arbuscule Development But Not the Maintenance of Arbuscule in Mycorrhizal Rice Roots.

Phosphorus (P) is a crucial nutrient for plant growth, but its availability to roots is limited in soil. Arbuscular mycorrhizal (AM) symbiosis is a promising strategy for improving plant P acquisition. However, P fertilizer reduces fungal colonization (P inhibition) and compromises mycorrhizal P uptake, warranting studies on the mechanistic basis of P inhibition. In this study, early morphological changes in P inhibition were identified in rice (Oryza sativa) using fungal cell wall staining and live-cell imaging of plant membranes that were associated with arbuscule life cycles. Arbuscule density decreased, and aberrant hyphal branching was observed in roots at 5 h after P treatment. Although new arbuscule development was severely inhibited, preformed arbuscules remained intact and longevity remained constant. P inhibition was accelerated in the rice pt11-1 mutant, which lacks P uptake from arbuscule branches, suggesting that mature arbuscules are stabilized by the symbiotic P transporter under high P condition. Moreover, P treatment led to increases in the number of vesicles, in which lipid droplets accumulated and then decreased within a few days. The development of new arbuscules resumed within by 2 d. Our data established that P strongly and temporarily inhibits new arbuscule development, but not intraradical accommodation of AM fungi.

Growth Properties and Biomass Production in the Hybrid C4 Crop Sorghum bicolor.

Hybrid vigor (heterosis) has been used as a breeding technique for crop improvement to achieve enhanced biomass production, but the physiological mechanisms underlying heterosis remain poorly understood. In this study, to find a clue to the enhancement of biomass production by heterosis, we systemically evaluated the effect of heterosis on the growth rate and photosynthetic efficiency in sorghum hybrid [Sorghum bicolor (L.) Moench cv. Tentaka] and its parental lines (restorer line and maintainer line). The final biomass of Tentaka was 10-14 times greater than that of the parental lines grown in an experimental field, but the relative growth rate during the vegetative growth stage did not differ. Tentaka exhibited a relatively enlarged leaf area with lower leaf nitrogen content per leaf area (Narea). When the plants were grown hydroponically at different N levels, daily CO2 assimilation per leaf area (A) increased with Narea, and the ratio of A to Narea (N-use efficiency) was higher in the plants grown at low N levels but not different between Tentaka and the parental lines. The relationships between the CO2 assimilation rate, the amounts of photosynthetic enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase, Chl and Narea did not differ between Tentaka and the parental lines. Thus, Tentaka tended to exhibit enlargement of leaf area with lower N content, leading to a higher N-use efficiency for CO2 assimilation, but the photosynthetic properties did not differ. The greater biomass in Tentaka was mainly due to the prolonged vegetative growth period.