Robotized indoor phenotyping allows genomic prediction of adaptive traits in the field.

Breeding for resilience to climate change requires considering adaptive traits such as plant architecture, stomatal conductance and growth, beyond the current selection for yield. Robotized indoor phenotyping allows measuring such traits at high throughput for speed breeding, but is often considered as non-relevant for field conditions. Here, we show that maize adaptive traits can be inferred in different fields, based on genotypic values obtained indoor and on environmental conditions in each considered field. The modelling of environmental effects allows translation from indoor to fields, but also from one field to another field. Furthermore, genotypic values of considered traits match between indoor and field conditions. Genomic prediction results in adequate ranking of genotypes for the tested traits, although with lesser precision for elite varieties presenting reduced phenotypic variability. Hence, it distinguishes genotypes with high or low values for adaptive traits, conferring either spender or conservative strategies for water use under future climates.

Precise control of water stress in the field reveals different response thresholds for forage yield and digestibility of maize hybrids.

Introduction: With dwindling global freshwater supplies and increasing water stress, agriculture is coming under increasing pressure to reduce water use. Plant breeding requires high analytical capabilities. For this reason, near-infrared spectroscopy (NIRS) has been used to develop prediction equations for whole-plant samples, particularly for predicting dry matter digestibility, which has a major impact on the energy value of forage maize hybrids and is required for inclusion in the official French catalogue. Although the historical NIRS equations have long been used routinely in seed company breeding programmes, they do not predict all variables with the same accuracy. In addition, little is known about how accurate their predictions are under different water stress-environments. Methods: Here, we examined the effects of water stress and stress intensity on agronomic, biochemical, and NIRS predictive values in a set of 13 modern S0-S1 forage maize hybrids under four different environmental conditions resulting from the combination of a northern and southern location and two monitored water stress levels in the south. Results: First, we compared the reliability of NIRS predictions for basic forage quality traits obtained using the historical NIRS predictive equations and the new equations we recently developed. We found that NIRS predicted values were affected to varying degrees by environmental conditions. We also showed that forage yield gradually decreased as a function of water stress, whereas both dry matter and cell wall digestibilities increased regardless of the intensity of water stress, with variability among the tested varieties decreasing under the most stressed conditions. Discussion: By combining forage yield and dry matter digestibility, we were able to quantify digestible yield and identify varieties with different strategies for coping with water stress, raising the exciting possibility that important potential selection targets still exist. Finally, from a farmer's perspective, we were able to show that late silage harvest has no effect on dry matter digestibility and that moderate water stress does not necessarily result in a loss of digestible yield.

Natural Variation in Vitamin B1 and Vitamin B6 Contents in Rice Germplasm.

Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger-a global health problem affecting approximately 2 billion people. Vitamin B1 (thiamine) and vitamin B6 (pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm-a poor source of several micronutrients leading to deficiencies when consumed monotonously-have led to only minimal improvements in vitamin B1 and B6 contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B1 and vitamin B6 contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B1 and B6 contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B1 and B6 biosynthesis de novo enzymes (THIC for vitamin B1, PDX1.3a-c and PDX2 for vitamin B6) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except for THIC expression in leaves that was positively correlated with total vitamin B1 contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B1 and B6 biosynthesis in rice.

Enhancement of vitamin B6 levels in rice expressing Arabidopsis vitamin B6 biosynthesis de novo genes.

Vitamin B6 (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B6 content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B6 in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B6 biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B6 levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B6 biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B6 in leaves (up to 28.3-fold) and roots (up to 12-fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B6 did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B6 content could also be achieved in rice seeds (up to 3.1-fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B6 -enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.

Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots.

AIM: We report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots. BACKGROUND: VIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function. RESULTS: We developed an African cassava mosaic virus-based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots. CONCLUSIONS: The African cassava mosaic virus-based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.

Vitamin B1 diversity and characterization of biosynthesis genes in cassava.

Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions. HPLC analyses of B1 vitamers revealed distinct profiles in cassava leaves and storage roots, with nearly equal relative levels of thiamin pyrophosphate and thiamin monophosphate in leaves, but mostly thiamin pyrophosphate in storage roots. Unusually, the cassava genome has two genes encoding the 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, THIC (MeTHIC1 and MeTHIC2), both of which carry a riboswitch in the 3'-UTR, as well as the adenylated thiazole synthase, THI1 (MeTHI1a and MeTHI1b). The THIC and THI1 genes are expressed at very low levels in storage roots compared with the accumulation of vitamin B1, indicating only limited biosynthesis de novo therein. In leaves, vitamin B1 content is negatively correlated with THIC and THI1 expression levels, suggesting post-transcriptional regulation of THIC by the riboswitch present in the 3'-UTR of the THIC mRNA and regulation of THI1 by promoter activity or alternative post-transcriptional mechanisms.

Rationalising vitamin B6 biofortification in crop plants.

Vitamin B6 encompasses a group of related compounds (vitamers) that can only be biosynthesised de novo by plants and microorganisms. Enzymatic cofactor and antioxidant functions for vitamin B6 are established in all kingdoms. Human vitamin B6 dietary insufficiency or genetic defects in B6 vitamer interconversion result in various neurological and inflammatory pathologies with several populations at-risk or marginal for vitamin B6 status. Three (rice, wheat and cassava) of the world's top five staple crops do not meet the recommended dietary allowance for vitamin B6, when consumed as a major proportion of the diet. In addition, controlled enhancement of the appropriate B6 vitamer in crops has the potential to confer stress resistance. Thus, crop biofortification strategies represent an opportunity to reduce the risk of deficiency in populations with limited diet diversity and quality, as well as improving stress tolerance.