John E. Sheehy

Using C4 photosynthesis to increase the yield of rice-rationale and feasibility.

90% of the worlds rice is grown and consumed in Asia, with each hectare of rice-producing land providing food for 27 people. By 2050, because of population growth and increasing urbanisation, each remaining hectare will have to feed at least 43 people. This means that yields must be increased by at least 50% over the next 40 years to prevent mass malnutrition for the 700 million Asians that currently rely on rice for more than 60% of their daily calorific intake. Since predictive models suggest that yield increases of this magnitude can only be achieved by improving photosynthesis, and because evolution has increased photosynthetic efficiency by 50% in the form of the C4 pathway, one solution is to generate C4 rice. However, this is an ambitious goal that requires proof of concept before any major investment of time and money. Here, we discuss approaches that should allow proof of concept to be tested.

Management of phosphorus, potassium, and sulfur in intensive, irrigated lowland rice

Abstract Management of soil phosphorus (P), potassium (K) and sulfur (S) resources in intensive, irrigated rice systems has received less attention than increasing cropping intensity and yields with new cultivars, irrigation, and fertilizer N. Crop requirements, input-output balance, and soil supplying capacity of P, K and S in irrigated lowland rice are reviewed. Based on projected rice production requirements, we estimate that the total annual nutrient demand for irrigated rice will be about 9 to 13 × 106 t N, 9 to 15 × 106 t K, 1.2 to 2.4 × 106 t P and 0.9 to 1.5 × 106 t S in 2025, amounts that represent an increase of 65 to 70% above 1990 requirements. At present, negative K balances are widespread and K deficiency has become a constraint to increasing yields, even on heavy-textured lowland soils with high inherent fertility. Because opportunities are limited for breeding cultivars that acquire more P, K or S from soil or have higher internal nutrient-use efficiencies, long-term management strategies must focus on maintaining adequate nutrient balances in the topsoil layer. Interactions among nutrients have a large influence on physiological and agronomic efficiency that result from nutrient applications. Strategies that only aim at increasing P or K application rates without considering the indigenous supply from soil reserves are inefficient; they may not sustain yield increases to meet rice demand. Little improvement in fertilizer use efficiency can be expected from the present system of providing blanket recommendations for a given production domain. Instead, site-specific nutrient-management approaches will be needed to accommodate the tremendous variability in indigenous nutrient supply found in the irrigated lowlands of Asia.

Spikelet numbers, sink size and potential yield in rice

Abstract A new plant type (NPT) has been developed at the International Rice Research Institute (IRRI) using tropical japonica germplasm in an attempt to increase yields. In direct contrast to high yielding indicas, which have large numbers of tillers and relatively small panicles, the NPT has large panicles and low tiller numbers. To determine whether the NPT had a higher potential for spikelet production, and possibly yield, than an elite indica, the total number of juvenile spikelets (glumous flower primordia) was determined for plants grown in the field as a crop or widely spaced. The maximum number of juvenile spikelets produced by the temporal pattern of tillers in a hill was recorded at the time of late differentiation of each tiller’s panicle. The number was greater for spaced plants than crop plants and it decreased linearly with tiller age. For crop plants approximately one juvenile spikelet was produced per square centimeter of leaf area. In 1997, a large number of the juvenile spikelets were lost before harvest, that number increased significantly in the wetter year of 1999. To quantify potential yield and its reduction by the loss of spikelets between the juvenile and mature stages, the number of spikelets at harvest and the filled fraction (grains) was determined for each tiller and a simple empirical model of yield was constructed. The model predicted that if all of the juvenile spikelets were to be transformed into grains a potential yield more than double the actual yield would be produced in crops of high yielding indicas or the NPT, suggesting that source strength rather than sink size limited yield in this study. Differences in spikelet number at harvest could be offset by differences in individual grain weight and percentage filling of spikelets. Even in rice crops grown with large applications of fertilizer, at current atmospheric concentrations of CO2, yields are limited by the supply of resources necessary for the development and synthesis of the grain rather than sink capacity. The potential sink size of the crop is almost double that actually realized, even though the productive tillers of crop plants do not reach their full potential for spikelet production compared with spaced plants.

Relationship between tillering and leaf area index: quantifying critical leaf area index for tillering in rice

A field study was conducted at the International Rice Research Institute (IRRI), Philippines during the dry seasons of 1997 and 1998 under irrigated conditions. The objectives of this study were to quantify the critical leaf area index (LAI c ) at which tillering stops based on the relationship between tillering rate and LAI, and to determine the effect of nitrogen (N) on LAI c in irrigated rice ( Oryza sativa L.) crop. Results showed that the relative tillering rate (RTR) decreased exponentially as LAI increased at a given N input level. The coefficient of determination for the equation quantifying the RTR-LAI relationship ranged from 0·87 to 0·99. The relationship between RTR and LAI was affected by N input level, but not by planting density. The N input level had a significant effect on LAI c with a high N input level causing an increase in LAI c . Tillering stopped at LAI of 3·36 to 4·11 when N was not limiting. Under N limited conditions LAI c reduced to as low as 0·98. Transplanting spacing and number of seedlings per hill had little effect on LAI c . Results from this study suggest that LAI and plant N status are two major factors that influence tiller production in rice crops. The possibility that LAI influences tillering by changing light intensity and/or light quality at the base of the canopy where tiller buds and young tillers are located is discussed.

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 C(4) traits into C(3) plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C(4) 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 C(4) species from C(3) species. To identify genetic factors that specify C(4) leaf anatomy, we generated ethyl methanesulfonate- and γ-ray-mutagenized populations of the C(4) 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 F(2) 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 C(4) and C(3) leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.

A sorghum (Sorghum bicolor) mutant with altered carbon isotope ratio

Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium’s efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C) of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor) mutant with a low δ13C characteristic. A mutant (named Mut33) with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT). The back-cross (BC1F1) progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air) and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs) between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used in the transformation of C3 to C4 plants.

Increasing leaf vein density via mutagenesis in rice results in an enhanced rate of photosynthesis, smaller cell sizes and can reduce interveinal mesophyll cell number

Improvements to leaf photosynthetic rates of crops can be achieved by targeted manipulation of individual component processes, such as the activity and properties of RuBisCO or photoprotection. This study shows that simple forward genetic screens of mutant populations can also be used to rapidly generate photosynthesis variants that are useful for breeding. Increasing leaf vein density (concentration of vascular tissue per unit leaf area) has important implications for plant hydraulic properties and assimilate transport. It was an important step to improving photosynthetic rates in the evolution of both C3 and C4 species and is a foundation or prerequisite trait for C4 engineering in crops like rice (Oryza sativa). A previous high throughput screen identified five mutant rice lines (cv. IR64) with increased vein densities and associated narrower leaf widths (Feldman et al., 2014). Here, these high vein density rice variants were analyzed for properties related to photosynthesis. Two lines were identified as having significantly reduced mesophyll to bundle sheath cell number ratios. All five lines had 20% higher light saturated photosynthetic capacity per unit leaf area, higher maximum carboxylation rates, dark respiration rates and electron transport capacities. This was associated with no significant differences in leaf thickness, stomatal conductance or CO2 compensation point between mutants and the wild-type. The enhanced photosynthetic rate in these lines may be a result of increased RuBisCO and electron transport component amount and/or activity and/or enhanced transport of photoassimilates. We conclude that high vein density (associated with altered mesophyll cell length and number) is a trait that may confer increased photosynthetic efficiency without increased transpiration.