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.

Into the Shadows and Back into Sunlight: Photosynthesis in Fluctuating Light.

Photosynthesis is an important remaining opportunity for further improvement in the genetic yield potential of our major crops. Measurement, analysis, and improvement of leaf CO2 assimilation (A) have focused largely on photosynthetic rates under light-saturated steady-state conditions. However, in modern crop canopies of several leaf layers, light is rarely constant, and the majority of leaves experience marked light fluctuations throughout the day. It takes several minutes for photosynthesis to regain efficiency in both sun-shade and shade-sun transitions, costing a calculated 10-40% of potential crop CO2 assimilation. Transgenic manipulations to accelerate the adjustment in sun-shade transitions have already shown a substantial productivity increase in field trials. Here, we explore means to further accelerate these adjustments and minimize these losses through transgenic manipulation, gene editing, and exploitation of natural variation. Measurement andanalysis of photosynthesis in sun-shade and shade-sun transitions are explained. Factors limiting speeds of adjustment and how they could be modified to effect improved efficiency are reviewed, specifically nonphotochemical quenching (NPQ), Rubisco activation, and stomatal responses.

High sink strength prevents photosynthetic down-regulation in cassava grown at elevated CO2 concentration.

Cassava has the potential to alleviate food insecurity in many tropical regions, yet few breeding efforts to increase yield have been made. Improved photosynthetic efficiency in cassava has the potential to increase yields, but cassava roots must have sufficient sink strength to prevent carbohydrates from accumulating in leaf tissue and suppressing photosynthesis. Here, we grew eight farmer-preferred African cassava cultivars under free-air CO2 enrichment (FACE) to evaluate the sink strength of cassava roots when photosynthesis increases due to elevated CO2 concentrations ([CO2]). Relative to the ambient treatments, elevated [CO2] treatments increased fresh (+27%) and dry (+37%) root biomass, which was driven by an increase in photosynthesis (+31%) and the absence of photosynthetic down-regulation over the growing season. Moreover, intrinsic water use efficiency improved under elevated [CO2] conditions, while leaf protein content and leaf and root cyanide concentrations were not affected. Overall, these results suggest that higher cassava yields can be expected as atmospheric [CO2] increases over the coming decades. However, there were cultivar differences in the partitioning of resources to roots versus above-grown biomass; thus, the particular responses of each cultivar must be considered when selecting candidates for improvement.

Photosynthesis across African cassava germplasm is limited by Rubisco and mesophyll conductance at steady state, but by stomatal conductance in fluctuating light.

Sub-Saharan Africa is projected to see a 55% increase in food demand by 2035, where cassava (Manihot esculenta) is the most widely planted crop and a major calorie source. Yet, cassava yield in this region has not increased significantly for 13 yr. Improvement of genetic yield potential, the basis of the first Green Revolution, could be realized by improving photosynthetic efficiency. First, the factors limiting photosynthesis and their genetic variability within extant germplasm must be understood. Biochemical and diffusive limitations to leaf photosynthetic CO2 uptake under steady state and fluctuating light in 13 farm-preferred and high-yielding African cultivars were analyzed. A cassava leaf metabolic model was developed to quantify the value of overcoming limitations to leaf photosynthesis. At steady state, in vivo Rubisco activity and mesophyll conductance accounted for 84% of the limitation. Under nonsteady-state conditions of shade to sun transition, stomatal conductance was the major limitation, resulting in an estimated 13% and 5% losses in CO2 uptake and water use efficiency, across a diurnal period. Triose phosphate utilization, although sufficient to support observed rates, would limit improvement in leaf photosynthesis to 33%, unless improved itself. The variation of carbon assimilation among cultivars was three times greater under nonsteady state compared to steady state, pinpointing important overlooked breeding targets for improved photosynthetic efficiency in cassava.

Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots.

BACKGROUND AND AIMS: Cell wall disassembly occurs naturally in plants by the action of several glycosyl-hydrolases during different developmental processes such as lysigenous and constitutive aerenchyma formation in sugarcane roots. Wall degradation has been reported in aerenchyma development in different species, but little is known about the action of glycosyl-hydrolases in this process. METHODS: In this work, gene expression, protein levels and enzymatic activity of cell wall hydrolases were assessed. Since aerenchyma formation is constitutive in sugarcane roots, they were assessed in segments corresponding to the first 5 cm from the root tip where aerenchyma develops. KEY RESULTS: Our results indicate that the wall degradation starts with a partial attack on pectins (by acetyl esterases, endopolygalacturonases, ß-galactosidases and α-arabinofuranosidases) followed by the action of ß-glucan-/callose-hydrolysing enzymes. At the same time, there are modifications in arabinoxylan (by α-arabinofuranosidases), xyloglucan (by XTH), xyloglucan-cellulose interactions (by expansins) and partial hydrolysis of cellulose. Saccharification revealed that access to the cell wall varies among segments, consistent with an increase in recalcitrance and composite formation during aerenchyma development. CONCLUSION: Our findings corroborate the hypothesis that hydrolases are synchronically synthesized, leading to cell wall modifications that are modulated by the fine structure of cell wall polymers during aerenchyma formation in the cortex of sugarcane roots.

The control of endopolygalacturonase expression by the sugarcane RAV transcription factor during aerenchyma formation.

The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing ß-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

BSD2 is a Rubisco-specific assembly chaperone, forms intermediary hetero-oligomeric complexes, and is nonlimiting to growth in tobacco.

The folding and assembly of Rubisco large and small subunits into L8 S8 holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco-BSD2 assembly complexes in the model C3 plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m2 ) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L8 S8 pool. RNAi-silencing BSD2 production in transplastomic tobacco producing bacterial L2 Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi-bsd2 alleles into wild-type tobacco however impaired L8 S8 Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2-silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.

Toward improving photosynthesis in cassava: Characterizing photosynthetic limitations in four current African cultivars.

Despite the vast importance of cassava (Manihot esculenta Crantz) for smallholder farmers in Africa, yields per unit land area have not increased over the past 55 years. Genetic engineering or breeding for increased photosynthetic efficiency may represent a new approach. This requires the understanding of limitations to photosynthesis within existing germplasm. Here, leaf photosynthetic gas exchange, leaf carbon and nitrogen content, and nonstructural carbohydrates content and growth were analyzed in four high-yielding and farm-preferred African cultivars: two landraces (TME 7, TME 419) and two improved lines (TMS 98/0581 and TMS 30572). Surprisingly, the two landraces had, on average, 18% higher light-saturating leaf CO 2 uptake (Asat) than the improved lines due to higher maximum apparent carboxylation rates of Rubisco carboxylation (Vcmax) and regeneration of ribulose-1,5-biphosphate expressed as electron transport rate (Jmax). TME 419 also showed a greater intrinsic water use efficiency. Except for the cultivar TMS 30572, photosynthesis in cassava showed a triose phosphate utilization (TPU) limitation at high intercellular [CO 2]. The capacity for TPU in the leaf would not limit photosynthesis rates under current conditions, but without modification would be a barrier to increasing photosynthetic efficiency to levels predicted possible in this crop. The lower capacity of the lines improved through breeding, may perhaps reflect the predominant need, until now, in cassava breeding for improved disease and pest resistance. However, the availability today of equipment for high-throughput screening of photosynthetic capacity provides a means to select for maintenance or improvement of photosynthetic capacity while also selecting for pest and disease resistance.

Diurnal variation in gas exchange and nonstructural carbohydrates throughout sugarcane development.

Photosynthesis and growth are dependent on environmental conditions and plant developmental stages. However, it is still not clear how the environment and development influence the diurnal dynamics of nonstructural carbohydrates production and how they affect growth. This is particularly the case of C4 plants such as sugarcane (Saccharum spp.). Aiming to understand the dynamics of leaf gas exchange and nonstructural carbohydrates accumulation in different organs during diurnal cycles across the developmental stages, we evaluated these parameters in sugarcane plants in a 12-month field experiment. Our results show that during the first 3 months of development, light and vapour pressure deficit (VPD) were the primary drivers of photosynthesis, stomatal conductance and growth. After 6 months, in addition to light and VPD, drought, carbohydrate accumulation and the mechanisms possibly associated with water status maintenance were also likely to play a role in gas exchange and growth regulation. Carbohydrates vary throughout the day in all organs until Month 9, consistent with their use for growth during the night. At 12 months, sucrose is accumulated in all organs and starch had accumulated in leaves without any diurnal variation. Understanding of how photosynthesis and the dynamics of carbohydrates are controlled might lead to strategies that could increase sugarcane's biomass production.

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia.

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia.

The Role of Sink Strength and Nitrogen Availability in the Down-Regulation of Photosynthetic Capacity in Field-Grown Nicotiana tabacum L. at Elevated CO2 Concentration.

Down-regulation of photosynthesis is among the most common responses observed in C3 plants grown under elevated atmospheric CO2 concentration ([CO2]). Down-regulation is often attributed to an insufficient capacity of sink organs to use or store the increased carbohydrate production that results from the stimulation of photosynthesis by elevated [CO2]. Down-regulation can be accentuated by inadequate nitrogen (N) supply, which may limit sink development. While there is strong evidence for down-regulation of photosynthesis at elevated [CO2] in enclosure studies most often involving potted plants, there is little evidence for this when [CO2] is elevated fully under open-air field treatment conditions. To assess the importance of sink strength on the down-regulation of photosynthesis and on the potential of N to mitigate this down-regulation under agriculturally relevant field conditions, two tobacco cultivars (Nicotiana tabacum L. cv. Petit Havana; cv. Mammoth) of strongly contrasting ability to produce the major sink of this crop, leaves, were grown under ambient and elevated [CO2] and with two different N additions in a free air [CO2] (FACE) facility. Photosynthetic down-regulation at elevated [CO2] reached only 9% in cv. Mammoth late in the season likely reflecting sustained sink strength of the rapidly growing plant whereas down-regulation in cv. Petit Havana reached 25%. Increased N supply partially mitigated down-regulation of photosynthesis in cv. Petit Havana and this mitigation was dependent on plant developmental stage. Overall, these field results were consistent with the hypothesis that sustained sink strength, that is the ability to utilize photosynthate, and adequate N supply will allow C3 crops in the field to maintain enhanced photosynthesis and therefore productivity as [CO2] continues to rise.

Rooting for cassava: insights into photosynthesis and associated physiology as a route to improve yield potential.

Contents 50 I. 50 II. 52 III. 54 IV. 55 V. 57 VI. 57 VII. 59 60 References 61 SUMMARY: As a consequence of an increase in world population, food demand is expected to grow by up to 110% in the next 30-35 yr. The population of sub-Saharan Africa is projected to increase by > 120%. In this region, cassava (Manihot esculenta) is the second most important source of calories and contributes c. 30% of the daily calorie requirements per person. Despite its importance, the average yield of cassava in Africa has not increased significantly since 1961. An evaluation of modern cultivars of cassava showed that the interception efficiency (ɛi ) of photosynthetically active radiation (PAR) and the efficiency of conversion of that intercepted PAR (ɛc ) are major opportunities for genetic improvement of the yield potential. This review examines what is known of the physiological processes underlying productivity in cassava and seeks to provide some strategies and directions toward yield improvement through genetic alterations to physiology to increase ɛi and ɛc . Possible physiological limitations, as well as environmental constraints, are discussed.

Changes in Whole-Plant Metabolism during the Grain-Filling Stage in Sorghum Grown under Elevated CO2 and Drought.

Projections indicate an elevation of the atmospheric CO2 concentration ([CO2]) concomitant with an intensification of drought for this century, increasing the challenges to food security. On the one hand, drought is a main environmental factor responsible for decreasing crop productivity and grain quality, especially when occurring during the grain-filling stage. On the other hand, elevated [CO2] is predicted to mitigate some of the negative effects of drought. Sorghum (Sorghum bicolor) is a C4 grass that has important economical and nutritional values in many parts of the world. Although the impact of elevated [CO2] and drought in photosynthesis and growth has been well documented for sorghum, the effects of the combination of these two environmental factors on plant metabolism have yet to be determined. To address this question, sorghum plants (cv BRS 330) were grown and monitored at ambient (400 µmol mol(-1)) or elevated (800 µmol mol(-1)) [CO2] for 120 d and subjected to drought during the grain-filling stage. Leaf photosynthesis, respiration, and stomatal conductance were measured at 90 and 120 d after planting, and plant organs (leaves, culm, roots, prop roots, and grains) were harvested. Finally, biochemical composition and intracellular metabolites were assessed for each organ. As expected, elevated [CO2] reduced the stomatal conductance, which preserved soil moisture and plant fitness under drought. Interestingly, the whole-plant metabolism was adjusted and protein content in grains was improved by 60% in sorghum grown under elevated [CO2].

How endogenous plant cell-wall degradation mechanisms can help achieve higher efficiency in saccharification of biomass.

Cell-wall recalcitrance to hydrolysis still represents one of the major bottlenecks for second-generation bioethanol production. This occurs despite the development of pre-treatments, the prospect of new enzymes, and the production of transgenic plants with less-recalcitrant cell walls. Recalcitrance, which is the intrinsic resistance to breakdown imposed by polymer assembly, is the result of inherent limitations in its three domains. These consist of: (i) porosity, associated with a pectin matrix impairing trafficking through the wall; (ii) the glycomic code, which refers to the fine-structural emergent complexity of cell-wall polymers that are unique to cells, tissues, and species; and (iii) cellulose crystallinity, which refers to the organization in micro- and/or macrofibrils. One way to circumvent recalcitrance could be by following cell-wall hydrolysis strategies underlying plant endogenous mechanisms that are optimized to precisely modify cell walls in planta. Thus, the cell-wall degradation that occurs during fruit ripening, abscission, storage cell-wall mobilization, and aerenchyma formation are reviewed in order to highlight how plants deal with recalcitrance and which are the routes to couple prospective enzymes and cocktail designs with cell-wall features. The manipulation of key enzyme levels in planta can help achieving biologically pre-treated walls (i.e. less recalcitrant) before plants are harvested for bioethanol production. This may be helpful in decreasing the costs associated with producing bioethanol from biomass.

How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.

The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.