Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.     

C4 anatomy can evolve via a single developmental change

C₄ photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C₄ evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C₄ functionality. Here, we quantify the anatomical changes accompanying the transition between non‐C₄ and C₄ phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C₄ individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C₄ function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C₄ leaf anatomy, creating an evolutionary entry point to complex C₄ syndromes that can become more specialised.     

Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae)

Background and Aims

Leaf venation in many C₄ species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C₄ photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C₄ photosynthesis evolution. Development of C₄ vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development.

Methods

Cleared and sectioned tissues of phylogenetically basal C₃ Flaveria robusta and more derived C₄ Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves.

Key Results

In mature leaves, major vein density did not differ between C₃ and C₄ Flaveria species, whereas minor veins were denser in C₄ species than in C₃ species. The developmental study showed that both major and minor vein patterning in leaves of C₃ and C₄ species were initiated at comparable stages (based on leaf length). An additional vein order in the C₄ species was observed during initiation of the higher order minor veins compared with the C₃ species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C₄ species than in C₃ species.

Conclusions

Leaf vein pattern characteristic to C₄ Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C₃ species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C₄ species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C₄ species.Key words: Bundle sheath, C₄ photosynthesis evolution, Flaveria, heterochrony, leaf development, mesophyll, vein density, vein pattern formation     

Photosynthetic characteristics of hybrid rice with phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase gene

High level of phosphoenolpyruvate carboxylase (PEPC) gene was stably inherited and transferred from the male parent, PEPC transgenic rice, into a female parent, japonica rice cv. 9516. Relative to the female parent, the produced JAAS45 pollen lines exhibited high PEPC activity (17-fold increase) and also higher photosynthetic rates (about 36 %-fold increase). The JAAS45 pollen lines were more tolerant to photoinhibition and to photo-oxidative stress. Furthermore, JAAS45 pollen lines, as well as their male parent, were tested to exhibit a limiting C₄ cycle by feeding with exogenous C₄ primary products such as oxaloacetate (OAA). Thus the PEPC gene and photosynthetic characteristics of PEPC transgenic rice could be stably transferred to the hybrid progenies, which might open a new breeding approach to the integration of conventional hybridization and biological technology. An erratum to this article is available at .     

Comparative Anatomy and Morphology of Leaves between C3 and C4 Species in Panicum

Anatomical and morphological structures of leaf blades werecompared between C₃ and C₄ species in Panicum. Inter-specificvariation of stomatal density, longitudinal vein density andmesophyll thickness was highly correlative either plus or minuswithin respective groups. The two groups could not be distinguishedby a single character, since the variation ranges overlappedeach other. However, the quantitative relations between veindensity and the other two characters differentiated the twogroups well. In C₃, stomatal density seemed to be a primaryfactor for regulating water balance, while in C₄ vein systemwas considered to be important for the regulation. The specieswith intermediate photosynthesis behaved similar to the C₃ species.In the C₃ group, correlative variation was observed betweenleaf width, leaf angle and the three characters mentioned above.Variation of light-receiving area due to the changes of widthand angle of leaf blades was considered to be one of the adaptivestrategies of this group. Increase of light-receiving area wasin connection with the thinning of leaves. On the other hand,in the C₄ correlations between length, width and angle of leaveswere low. Such loose character correlation may be achieved byits efficiency of CO₂ utilization and its well developed veinsystems. Besides, NAD-me type species tended to have relativelylower stomatal and vein densities as compared with the otherdecarboxylation types in this group. Panicum, photosynthesis, C₃, C₄, decarboxylation types, leaf, stomata, vein     

C4 photosynthesis in C3 rice: a theoretical analysis of biochemical and anatomical factors

Engineering C₄ photosynthesis into rice has been considered a promising strategy to increase photosynthesis and yield. A question that remains to be answered is whether expressing a C₄ metabolic cycle into a C₃ leaf structure and without removing the C₃ background metabolism improves photosynthetic efficiency. To explore this question, we developed a 3D reaction diffusion model of bundle‐sheath and connected mesophyll cells in a C₃ rice leaf. Our results show that integrating a C₄ metabolic pathway into rice leaves with a C₃ metabolism and mesophyll structure may lead to an improved photosynthesis under current ambient CO₂ concentration. We analysed a number of physiological factors that influence the CO₂ uptake rate, which include the chloroplast surface area exposed to intercellular air space, bundle‐sheath cell wall thickness, bundle‐sheath chloroplast envelope permeability, Rubisco concentration and the energy partitioning between C₃ and C₄ cycles. Among these, partitioning of energy between C₃ and C₄ photosynthesis and the partitioning of Rubisco between mesophyll and bundle‐sheath cells are decisive factors controlling photosynthetic efficiency in an engineered C₃–C₄ leaf. The implications of the results for the sequence of C₄ evolution are also discussed.     

Gene actions of QTLs affecting several agronomic traits resolved in a recombinant inbred rice population and two backcross populations

To understand the types of gene action controlling seven quantitative traits in rice, we carried out quantitative trait locus (QTL) mapping in order to distinguish between the main-effect QTLs (M-QTLs) and digenic epistatic QTLs (E-QTLs) responsible for the trait performance of 254 recombinant inbred lines (RILs) from rice varieties Lemont/Teqing and two backcross hybrid (BCF₁) populations derived from these RILs. We identified 44 M-QTL and 95 E-QTL pairs in the RI and BCF₁ populations as having significant effects on the mean values and mid-parental heterosis of heading date, plant height, flag leaf length, flag leaf width, panicle length, spikelet number and spikelet fertility. The E-QTLs detected collectively explained a larger portion of the total phenotypic variation than the M-QTLs in both the RI and BCF₁ populations. In both BCF₁ populations, over-dominant (or under-dominant) loci were more important than additive and complete or partially dominant loci for M-QTLs and E-QTL pairs, thereby supporting prior findings that overdominance resulting from epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice.     

Adaptation potential of photosynthesis in wheat cultivars with a capability of leaf rolling under high temperature conditions

For the first time, the adaptive role of the rolling leaf trait for tolerance of wheat plants (Triticum aestivum L.) to the main factor of drought, high temperature, was demonstrated. Cv. Otan with high degree of this trait expression was more tolerant to temperature stress (40°C, 4 h during 2 days (2h/day)). Changes in parameters of chlorophyll fluorescence, F _v/F _m and R _Fd690, suggest that cv. Otan was tolerant to inhibition of photochemical activities of photosystem II (PSII) and photosystem I (PSI). Furthermore, high temperature had no effect on the rate of net photosynthesis (P _N) in cv. Otan, although it decreased this parameter in the other wheat cultivars. The main factors, which provid for this tolerance, were adaptation of the photosynthetic pigment system by active accumulation of carotenoids, more stable structural organization of PSII and PSI, and their high photosynthetic activities, as well as efficient stomatal regulation of transpiration and supplying of mesophyll cells with CO₂. It is hypothesized that the physiological role of the rolling leaf trait is the maintenance of adaptation potential by increasing the efficiency of water metabolism in the flag leaves of wheat under high temperature.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Partial nitrate nutrition amends photosynthetic characteristics in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. <Emphasis Type="Italic">japonica</Emphasis>) differing in nitrogen use efficiency

Partial nitrate nutrition (PNN) was found to improve rice (Oryza sativa L. var. japonica) growth. However, how PNN is related to photosynthesis in rice cultivars with different nitrogen use efficiency (NUE) is still not clear. Two rice cultivars, Nanguang (high NUE) and Elio (low NUE), were grown under sole NH₄ ⁺ and PNN at a total nitrogen concentration of 2.86 mM. The dry weight, leaf area, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and gas exchange parameters were measured. Nitrogen and Rubisco contents in the newly expanded leaves of cv. Nanguang were similar to those of cv. Elio when only NH₄ ⁺ was supplemented in the nutrient solution. However, in cv. Nanguang, nitrogen and Rubisco contents increased under PNN than under sole NH₄ ⁺ nutrition. Higher nitrogen and Rubisco contents were recorded in cv. Nanguang than in cv. Elio under PNN. The ratio of carboxylation efficiency (CE) to Rubisco content in cv. Nanguang was 11 and 14% higher than that in cv. Elio under NH₄ ⁺ and PNN, respectively. CE was 14% higher in cv. Nanguang than that in cv. Elio. The results suggest that PNN causes an increase in photosynthesis in cv. Nanguang. It is concluded that differences in Rubisco activity, rather than stomatal limitation, are responsible for the differences in photosynthesis between the two cultivars. The presence of nitrate increases Rubisco content in rice with a high NUE, which leads to faster biomass accumulation at later growth stages.     

Transgenic Rice Plants Expressing Bacillus subtilis Protoporphyrinogen Oxidase Gene Show Low Herbicide Oxyfluorfen Resistance

Transgenic rice plants harbouring Bacillus subtilis protoporphyrinogen oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T₁ generation by Southern and mRNA expression in T₂ generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice

Leaf morphology is an important agronomic trait in rice breeding. We isolated three allelic mutants of NARROW AND ROLLED LEAF 1 (nrl1) which showed phenotypes of reduced leaf width and semi-rolled leaves and different degrees of dwarfism. Microscopic analysis indicated that the nrl1-1 mutant had fewer longitudinal veins and smaller adaxial bulliform cells compared with the wild-type. The NRL1 gene was mapped to the chromosome 12 and encodes the cellulose synthase-like protein D4 (OsCslD4). Sequence analyses revealed single base substitutions in the three allelic mutants. Genetic complementation and over-expression of the OsCslD4 gene confirmed the identity of NRL1. The gene was expressed in all tested organs of rice at the heading stage and expression level was higher in vigorously growing organs, such as roots, sheaths and panicles than in elsewhere. In the mutant leaves, however, the expression level was lower than that in the wild-type. We conclude that OsCslD4 encoded by NRL1 plays a critical role in leaf morphogenesis and vegetative development in rice.     

Trait differences between grass species along a climatic gradient in South and North America

Question: Are trait differences between grasses along a gradient related to climatic variables and/or photosynthetic pathway? Location: Temperate grassland areas of South and North America. Methods: In a common garden experiment, we cultivated C₃ and C₄ grasses from grasslands under different climatic conditions, and we measured a set of 12 plant traits related to size and resource capture and utilization. We described (1) interspecific plant trait differences along a climatic gradient defined by the precipitation and temperature at the location where each species is dominant and (2) the association between those plant trait differences and the photosynthetic pathway of the species. Results: Trait differences between grasses were related to the precipitation at the area where each species is dominant, and to the photosynthetic pathway of the species. Leaf length, leaf width, plant height, leaf area per tiller, specific leaf area, leaf δ¹³C ratio, and nitrogen resorption efficiency increased while leaf dry matter content and nitrogen concentration in senesced leaves decreased as precipitation increased. A proportion of these changes along the gradient was related to the photosynthetic pathway because dominant grass species in cold areas with low precipitation are mainly C₃ and those from warm and wet areas are C₄. Conclusions: A previous worldwide analysis showed that traits of graminoid species measured in situ changed slightly along climatic gradients (< 10% variance explained). In contrast, under a common environment we observed that (1) grass traits changed strongly along a climatic gradient (30‐85% variance explained) and, (2) a proportion of those changes were related to the association between photosynthetic pathway of the species and precipitation.     

Mapping QTLs for defective female gametophyte development in an inter-subspecific cross in Oryza sativa L.

The embryo-sac is an essential structure for angiosperm reproduction. The cytological and genetic characterization of embryo-sac sterility was examined in a cross between Oryza sativa ssp. indica cv. ZYQ8 and ssp. japonica cultivar, JX17. The arrest of embryo-sac development was manifested following meiosis in the F₁ hybrid. When the megaspore carried the lethal genotype, the nucleus either failed to divide or divided only once, and the immature embryo-sac degenerated. Abortion of the embryo-sac in the indica-japonica hybrid background was not observed in their original parents, and an effect of cytoplasmic gene(s) on embryo-sac sterility in the reciprocal F₁ hybrids was not detected. Using a rice molecular linkage map based on a doubled haploid (DH) population from the cross of ZYQ8 /JX17, we mapped quantitative trait loci (QTLs) for the defective development of the female gametophyte in backcross progenies from the DH lines. The result demonstrated that a polygenic system is involved in both megagametogenesis and postzygotic isolation in inter-subspecific hybrid rice. Received: 4 May 2000 / Accepted: 20 September 2000     

Utilization of Stiff Culm Trait of Rice smos1 Mutant for Increased Lodging Resistance

Although the introduction of semi-dwarf trait into rice has led to improved lodging resistance making it capable of supporting high grain yield, lodging still remains a concern when attempting to further increase the grain yield of rice. However, improving the lodging resistance in rice by depending on the semi-dwarf trait alone is possible only up to a certain limit, beyond which other traits may be needed for reinforcement. To search for alternative traits relating to high lodging resistance, we identified 9 rice mutant lines possessing improved culm strength. To evaluate whether such lines can be useful for breeding lodging resistant rice, small organ size1 (smos1) mutant having increased lodging resistance but low tiller number and low grain yield, was chosen as a representative for a breeding trial. smos1 was crossed with ST-4 (from the Stock rice collection of Nagoya University Togo field #4), a cultivar with high tiller number and high grain yield, and from their progeny, LRC1 (lodging resistance candidate-1) was selected. Although the low tiller number trait of smos1 was not fully reversed in LRC1, this was compensated by an increase in grain weight per panicle, thereby resulting in high grain yield per plant. This important attribute of LRC1 was further enhanced by the improved lodging resistance trait inherited from smos1. Such improved lodging resistance in LRC1 and smos1 was revealed to be mainly due to increased culm diameter and culm thickness, which led to a high section modulus (SM) value, a parameter defining the physical strength of the culm. Since smos1 possesses high breaking-type lodging resistance which is different from semi-dwarf plants with high bending-type lodging resistance, an alternative approach of using thick culm lines for the creation of rice with increased lodging resistance is hereby proposed.     

Effect of soil drying on growth,biomass allocation and leaf gas exchange of two annual grass species

Influence of short-term water stress on plant growth and leaf gas exchange was studied simultaneously in a growth chamber experiment using two annual grass species differing in photosynthetic pathway type, plant architecture and phenology:Triticum aestivum L. cv. Katya-A-1 (C₃, a drought resistant wheat cultivar of erect growth) andTragus racemosus (L.) All. (C₄, a prostrate weed of warm semiarid areas). At the leaf level, gas exchange rates declined with decreasing soil water potential for both species in such a way that instantaneous photosynthetic water use efficiency (PWUE, mmol CO₂ assimilated per mol H₂O transpired) increased. At adequate water supply, the C₄ grass showed much lower stomatal conductance and higher PWUE than the C₃ species, but this difference disappeared at severe water stress when leaf gas exchange rates were similarly reduced for both species. However, by using soil water more sparingly, the C₄ species was able to assimilate under non-stressful conditions for a longer time than the C₃ wheat did. At the whole-plant level, decreasing water availability substantially reduced the relative growth rate (RGR) ofT. aestivum, while biomass partitioning changed in favour of root growth, so that the plant could exploit the limiting water resource more efficiently. The change in partitioning preceded the overall reduction of RGR and it was associated with increased biomass allocation to roots and less to leaves, as well as with a decrease in specific leaf area. Water saving byT. racemosus sufficiently postponed water stress effects on plant growth occurring only as a moderate reduction in leaf area enlargement. For unstressed vegetative plants, relative growth rate of the C₄ T. racemosus was only slightly higher than that of the C₃ T. aestivum, though it was achieved at a much lower water cost. The lack of difference in RGR was probably due to growth conditions being relatively suboptimal for the C₄ plant and also to a relatively large investment in stem tissues by the C₄ T. racemosus. Only 10% of the plant biomass was allocated to roots in the C₄ species while this was more than 30% for the C₃ wheat cultivar. These results emphasize the importance of water saving and high WUE of C₄ plants in maintaining growth under moderate water stress in comparison with C₃ species.     

Aquaporin plays an important role in mediating chloroplastic CO2 concentration under high‐N supply in rice (Oryza sativa) plants

Our previous studies demonstrated that chloroplastic CO₂ concentration (Cc) is not sufficient under high‐nitrogen (N) supply in rice plants. In this research, we studied how aquaporins‐ (AQPs) mediated Cc under different N‐supply levels. A hydroponic experiment was conducted in a greenhouse with three different N levels (low N, 0.71 mM; intermediate N, 2.86 mM; and high N, 7.14 mM) in a rice cultivar (Oryza sativa cv. Shanyou 63) and with an ospip1;1 mutant (Oryza sativa cv. Nipponbare). The photosynthetic nitrogen‐use efficiency (PNUE) decreased with increasing leaf‐N content. Under high‐N supply, the estimated Cc was significantly lower than the theoretical Cc and the specific Rubisco activity (carboxylation efficiency/Rubisco content, CE/Rubisco) decreased, because of a decrease of relative CO₂ diffusion conductance (total CO₂ diffusion conductance/leaf‐N content, g_t/N) in mesophyll cells. Real Time Quantitative PCR (Q‐RT‐PCR) showed that most OsPIP1s and OsPIP2s expression were downregulated under the high‐N supply. Furthermore, Cc and g_m decreased in the ospip1;1 mutant line compared with that of the wild‐type plant. It was concluded that under high‐N supply, the decreased PNUE was associated with non‐sufficient Cc, mediated by AQP in mesophyll conductance.