Response to Comments on "Soybean photosynthesis and crop yield is improved by accelerating recovery from photoprotection".

We recently demonstrated that accelerating the relaxation of nonphotochemical quenching leads to higher soybean photosynthetic efficiency and yield. In response, Sinclair et al. assert that improved photosynthesis cannot improve crop yields and that there is only one valid experimental design for proving a genetic improvement in yield. We explain here why neither assertion is valid.

Soybean photosynthesis and crop yield are improved by accelerating recovery from photoprotection.

Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. This protective dissipation continues after the leaf transitions to shade, reducing crop photosynthesis. A bioengineered acceleration of this adjustment increased photosynthetic efficiency and biomass in tobacco in the field. But could that also translate to increased yield in a food crop? Here we bioengineered the same change into soybean. In replicated field trials, photosynthetic efficiency in fluctuating light was higher and seed yield in five independent transformation events increased by up to 33%. Despite increased seed quantity, seed protein and oil content were unaltered. This validates increasing photosynthetic efficiency as a much needed strategy toward sustainably increasing crop yield in support of future global food security.

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

A drought-responsive rice amidohydrolase is the elusive plant guanine deaminase with the potential to modulate the epigenome.

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S-adenosyl-methionine (SAM) to xanthine pathway. SAM is converted to S-adenosyl-homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

Studies of rice Hd1 haplotypes worldwide reveal adaptation of flowering time to different environments.

Rice domestication/adaptation is a good model for studies of the development and spread of this important crop. Mutations that caused morphological and physiological change, followed by human selection/expansion, finally led to the improvement of phenotypes suitable for different kinds of environments. We used the sequence information for Heading date 1 (Hd1) gene to reveal the association between sequence changes and flowering phenotypes of rice in different regions. Seven loss-of-function hd1 haplotypes had been reported. By data-mining the genome sequencing information in the public domain, we discovered 3 other types. These loss-of-function allele haplotypes are present in subtropical and tropical regions, which indicates human selection. Some of these haplotypes are present locally. However, types 7 and 13 are present in more than one-third of the world's rice accessions, including landraces and modern varieties. In the present study, phylogenetic, allele network and selection pressure analyses revealed that these two haplotypes might have occurred early in Southeastern Asia and then were introgressed in many local landraces in nearby regions. We also demonstrate that these haplotypes are present in weedy rice populations, which again indicates that these alleles were present in rice cultivation for long time. In comparing the wild rice sequence information, these loss-of-function haplotypes occurred in agro but were not from wild rice.