Cassava shrunken-2 homolog MeAPL3 determines storage root starch and dry matter content and modulates storage root postharvest physiological deterioration.

KEY MESSAGE: Among the five cassava isoforms (MeAPL1-MeAPL5), MeAPL3 is responsible for determining storage root starch content. Degree of storage root postharvest physiological deterioration (PPD) is directly correlated with starch content. AGPase is heterotetramer composed of two small and two large subunits each coded by small gene families in higher plants. Studies in cassava (Manihot esculenta) identified and characterized five isoforms of Manihot esculenta ADP-glucose pyrophosphorylase large subunit (MeAPL1-MeAPL5) and employed virus induced gene silencing (VIGS) to show that MeAPL3 is the key isoform responsible for starch and dry matter accumulation in cassava storage roots. Silencing of MeAPL3 in cassava through stable transgenic lines resulted in plants displaying significant reduction in storage root starch and dry matter content (DMC) and induced a distinct phenotype associated with increased petiole/stem angle, resulting in a droopy leaf phenotype. Plants with reduced starch and DMC also displayed significantly reduced or no postharvest physiological deterioration (PPD) compared to controls and lines with high DMC and starch content. This provides strong evidence for direct relationships between starch/dry matter content and its role in PPD and canopy architecture traits in cassava.

Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch.

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A ß-carotene. In this study, ß-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxy-d-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 µg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%-90% of these carotenoids accumulated as all-trans-ß-carotene, the most nutritionally efficacious carotenoid. ß-Carotene-accumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in ß-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between ß-carotene and dry matter content, with reductions of 50%-60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between ß-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Author Correction: Biofortification of field-grown cassava by engineering expression of an iron transporter and ferritin.

In the version of this article initially published, a relevant work was not cited. The following sentence has been inserted following the sentence ending "Aspergillus phytase" in the third paragraph of the article: "Overexpression of AtIRT1, AtNAS1 and bean FERRITIN in rice resulted in 3.8-fold higher iron and 1.8-fold higher zinc concentrations than in the wild-type control12." A corresponding reference has been added: 12. Boonyaves, K., Wu, T. Y., Gruissem, W. & Bhullar, N. K. Enhanced grain iron levels in rice expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN gene cassette. Front. Plant Sci. 8, 130 (2017). The error has been corrected in the HTML and PDF versions of the article.

Biofortification of field-grown cassava by engineering expression of an iron transporter and ferritin.

Less than 10% of the estimated average requirement (EAR) for iron and zinc is provided by consumption of storage roots of the staple crop cassava (Manihot esculenta Crantz) in West African human populations. We used genetic engineering to improve mineral micronutrient concentrations in cassava. Overexpression of the Arabidopsis thaliana vacuolar iron transporter VIT1 in cassava accumulated three- to seven-times-higher levels of iron in transgenic storage roots than nontransgenic controls in confined field trials in Puerto Rico. Plants engineered to coexpress a mutated A. thaliana iron transporter (IRT1) and A. thaliana ferritin (FER1) accumulated iron levels 7-18 times higher and zinc levels 3-10 times higher than those in nontransgenic controls in the field. Growth parameters and storage-root yields were unaffected by transgenic fortification in our field data. Measures of retention and bioaccessibility of iron and zinc in processed transgenic cassava indicated that IRT1 + FER1 plants could provide 40-50% of the EAR for iron and 60-70% of the EAR for zinc in 1- to 6-year-old children and nonlactating, nonpregnant West African women.

Design of substrate label for steady state flux measurements in plant systems using the metabolic network of Brassica napus embryos.

Steady state metabolic flux analysis using (13)C labeled substrates is of growing importance in plant physiology and metabolic engineering. The quality of the flux estimates in (13)C metabolic flux analysis depend on the: (i) network structure; (ii) flux values; (iii) design of the labeling substrate; and (iv) label measurements performed. Whereas the first two parameters are facts of nature, the latter two are to some extent controlled by the experimenter, yet they have received little attention in most plant studies. Using the metabolic flux map of developing Brassica napus (Rapeseed) embryos, this study explores the value of optimal substrate label designs obtained with different statistical criteria and addresses the applicability of different optimal designs to biological questions. The results demonstrate the value of optimizing the choice of labeled substrates and show that substrate combinations commonly used in bacterial studies can be far from optimal for mapping fluxes in plant systems. The value of performing additional experiments and the inclusion of measurements is also evaluated.

A thioacidolysis method tailored for higher-throughput quantitative analysis of lignin monomers.

Thioacidolysis is a method used to measure the relative content of lignin monomers bound by ß-O-4 linkages. Current thioacidolysis methods are low-throughput as they require tedious steps for reaction product concentration prior to analysis using standard GC methods. A quantitative thioacidolysis method that is accessible with general laboratory equipment and uses a non-chlorinated organic solvent and is tailored for higher-throughput analysis is reported. The method utilizes lignin arylglycerol monomer standards for calibration, requires 1-2 mg of biomass per assay and has been quantified using fast-GC techniques including a Low Thermal Mass Modular Accelerated Column Heater (LTM MACH). Cumbersome steps, including standard purification, sample concentrating and drying have been eliminated to help aid in consecutive day-to-day analyses needed to sustain a high sample throughput for large screening experiments without the loss of quantitation accuracy. The method reported in this manuscript has been quantitatively validated against a commonly used thioacidolysis method and across two different research sites with three common biomass varieties to represent hardwoods, softwoods, and grasses.

Daylength and circadian effects on starch degradation and maltose metabolism.

Transitory starch is stored during the day inside chloroplasts and broken down at night for export. Maltose is the primary form of carbon export from chloroplasts at night. We investigated the influence of daylength and circadian rhythms on starch degradation and maltose metabolism. Starch breakdown was faster in plants of Arabidopsis (Arabidopsis thaliana) ecotype Wassilewskija growing in long days. Transcript levels of genes encoding enzymes involved in starch degradation and maltose metabolism showed a strong diurnal rhythm. Under altered photoperiods, the transcript levels and the rate of starch degradation changed within one day/night cycle. However, the amount of proteins involved in starch degradation was maintained relatively constant throughout the day/night cycle. To investigate whether the diurnal cycling of the transcript levels is only a response to light or is also regulated by a circadian clock, we measured the amount of messenger RNAs in Arabidopsis leaves under continuous light and continuous darkness. The expression of genes encoding starch degradation-related enzymes was under very strong circadian control in continuous light. Under continuous light, the amount of maltose also showed a strong endogenous rhythm close to 24 h, indicating that maltose metabolism is under circadian control. Light is necessary for the cycling of transcript levels and maltose levels. Under continuous darkness, these genes were barely expressed, and no cycling of maltose levels was observed.