Leaves are derived from heterotrophic meristem tissue that, at some point, must make the transition to autotrophy via the initiation of photosynthesis. However, the timing and spatial coordination of the molecular and cellular processes underpinning this switch are poorly characterized. Here, we report on the identification of a specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when photosynthetic competence is first established. Using a combined physiological and molecular approach, we show that elements of stomatal and vascular differentiation are coordinated with the onset of measurable light absorption for photosynthesis. Moreover, by exploring the response of the system to environmental perturbation, we show that the earliest stages of rice leaf development have significant plasticity with respect to elements of cellular differentiation of relevance for mature leaf photosynthetic performance. Finally, by performing an RNA sequencing analysis targeted at the early stages of rice leaf development, we uncover a palette of genes whose expression likely underpins the acquisition of photosynthetic capability. Our results identify the P3/P4 transition as a highly dynamic stage in rice leaf development when several processes for the initiation of photosynthetic competence are coordinated. As well as identifying gene targets for future manipulation of rice leaf structure/function, our data highlight a developmental window during which such manipulations are likely to be most effective.

Rice (Oryza sativa) is a water-intensive crop, and like other plants uses stomata to balance CO2 uptake with water-loss. To identify agronomic traits related to rice stomatal complexes, an anatomical screen of 64 Thai and 100 global rice cultivars was undertaken. Epidermal outgrowths called papillae were identified on the stomatal subsidiary cells of all cultivars. These were also detected on eight other species of the Oryza genus but not on the stomata of any other plant species we surveyed. Our rice screen identified two cultivars that had “mega-papillae” that were so large or abundant that their stomatal pores were partially occluded; Kalubala Vee had extra-large papillae, and Dharia had approximately twice the normal number of papillae. These were most accentuated on the flag leaves, but mega-papillae were also detectable on earlier forming leaves. Energy dispersive X-Ray spectrometry revealed that silicon is the major component of stomatal papillae. We studied the potential function(s) of mega-papillae by assessing gas exchange and pathogen infection rates. Under saturating light conditions, mega-papillae bearing cultivars had reduced stomatal conductance and their stomata were slower to close and re-open, but photosynthetic assimilation was not significantly affected. Assessment of an F3 hybrid population treated with Xanthomonas oryzae pv. oryzicola indicated that subsidiary cell mega-papillae may aid in preventing bacterial leaf streak infection. Our results highlight stomatal mega-papillae as a novel rice trait that influences gas exchange, stomatal dynamics, and defense against stomatal pathogens which we propose could benefit the performance of future rice crops.

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IntroductionThe anatomy of rice leaves is closely related to photosynthesis and grain yield. Therefore, exploring insight into the quantitative trait loci (QTLs) and alleles related to rice flag leaf anatomical and vein traits is vital for rice improvement.MethodsHere, we aimed to explore the genetic architecture of eight flag leaf traits using one single-locus model; mixed-linear model (MLM), and two multi-locus models; fixed and random model circulating probability unification (FarmCPU) and Bayesian information and linkage disequilibrium iteratively nested keyway (BLINK). We performed multi-model GWAS using 329 rice accessions of RDP1 with 700K single-nucleotide polymorphisms (SNPs) markers.ResultsThe phenotypic correlation results indicated that rice flag leaf thickness was strongly correlated with leaf mesophyll cells layer (ML) and thickness of both major and minor veins. All three models were able to identify several significant loci associated with the traits. MLM identified three non-synonymous SNPs near NARROW LEAF 1 (NAL1) in association with ML and the distance between minor veins (IVD) traits.DiscussionSeveral numbers of significant SNPs associated with known gene function in leaf development and yield traits were detected by multi-model GWAS performed in this study. Our findings indicate that flag leaf traits could be improved via molecular breeding and can be one of the targets in high-yield rice development.