Genetic Engineering and Genome Editing for Zinc Biofortification of Rice

Genetic Engineering and Genome Editing for Zinc Biofortification of Rice provides the first single-volume, comprehensive resource on genetic engineering approaches, including novel genome editing techniques, that are carried out in rice, a staple crop for much of the world’s population. Dietary zinc deficiency can lead to negative health outcomes, including increased risk of stunting, respiratory diseases, diarrhea, mortality during childhood, and preterm births in pregnancy. By providing a complete view of the need for zinc biofortification in rice, sections in this book discuss state-of-the-art scientific advances, and then go further, placing them in their proper scientific, regulatory and socioeconomic contexts.While zinc biofortification can be achieved through conventional breeding, genetic engineering and agronomic practices, this is the first reference to bring all the latest insights and understanding to a comprehensive resource that is based on real-world experience and targeted applications.

• Compiles the state-of-the-art information to allow fast-track understanding and application of zinc content improvement
• Discusses multiple strategic and methodology approaches
• Includes discussion of the socioeconomic implications of improved rice nutritional value

• Language: English
• Publisher: Academic Press
• Release date: Jun 23, 2023
• ISBN: 9780323854078

Book preview

Preface

Zinc (Zn) is an essential micronutrient required for a multitude of biological functions in all living beings. Zn plays a major role in catalytic activity of hundreds of enzymes and essential for the structural and functional stability of numerous proteins in the human body. Therefore, the importance of Zn in human growth, development, and health has been emphasized since ages. However, more than half of the world's population suffer from micronutrient deficiencies–related health problems; especially children, women and elderly people, poor and marginal populations living in rural areas of the developing world are highly vulnerable. Zn deficiency causes stunting, diarrhoea, reduced immunity, impaired cognitive development, reproductive health issues, etc. Moreover, recently, it has been shown that Zn-deficient people are highly prone to COVID-19. Zn malnutrition leads to millions of disability-adjusted life years (DALYs) in Asia, Africa, and Latin America. It has a significant bearing on the countries productivity and economic prosperity. Thus, addressing micronutrient malnutrition has been accorded a top priority at the global level by including it as one of the Sustainable Development Goals (SDGs) to be achieved by 2030. It aims to reduce pre- and postnatal mortalities, improved maternal health and nutrition, and zero hunger.

Zn deficiency is common in the populations that are entirely dependent on staple cereals for their nutritional needs. Even though nutrient supplementation, food fortification, and dietary diversification have been advocated, still the malnutrition persists to a larger extent in the developing world. Breeding staple crops with enhanced levels of bioavailable nutrients in their edible parts, also called as biofortification, is the most economical and a sustainable approach to address the malnutrition. Rice is among the priority crops for biofortification in Asia; it is being enriched with Fe, Zn, and vitamin A. There have been significant efforts over the last 2 decades to breed for nutritionally enriched rice varieties. Several conventionally bred high Zn rice varieties have been released for commercial cultivation in Asia, and first vitamin A–rich transgenic rice variety has been released in the Philippines.

There has been a significant improvement in our understanding on the genetic and molecular bases of metal homeostasis through identification of major QTLs and genes, dissecting their interactions and pathways in rice and other cereals. Genetic engineering (GE) and genome editing (GEd) technologies also called as next generation breeding Tools (NBTs) are being widely used to manipulate genes in a more precise and targeted way to develop rice varieties. Rice being a model crop has the standard, reliable and routinely useable protocols for efficient GE and GEd. In rice, multiple targeted genes including those related to mineral uptake, transport, and loading have been either engineered or edited in the genetic backgrounds of popular rice varieties. Several events with multifold increase in Fe and Zn without compromising yield have been developed, validated, and field tested. Efforts are also being made to stack Fe, Zn, and vitamin A. These results show the potential of GE and GEd technologies in developing biofortified rice varieties or rice varieties free of toxic mineral elements such as cadmium (Cd) and arsenic (As). Also with respect to regulatory science, intellectual property rights of GE or GEd, there is a tremendous progress, and some types of GEd are considered as non-Genetically Modified Organisms (GMOs) and treated on par with conventionally bred crop varieties in several countries. Economic feasibility studies have shown that NBTs are viable option for rice biofortification. There is also a growing acceptance and consumption of GE or GEd products globally. Therefore, it is necessary and timely to capture the recent advances in NBTs for crop improvement and especially for the rice nutritional improvement.

Biofortification scientists from the International Rice Research Institute (IRRI), Philippines, and the University of Pavia, Italy, have compiled a book titled Genetic Engineering and Genome Editing for Zinc Biofortification of Rice . It provides comprehensive information on various aspects of NBTs and their use in developing safer and nutritious rice varieties. This book consists of 14 chapters covering wide range of topics, that includes chapters on molecular mechanisms of Zn homeostasis and the cross-link between Fe and Zn biofortification, characterization of mineral accumulation using imaging technologies that provides basis for molecular breeding, and functional characterization. There are chapters dealing with detailed protocols for GE and GEd and stages in development of products using these tools. GEd is a cutting edge technology becoming hugely popular for genetic manipulations, so there is a chapter providing updated information on genome editing. Metal transporters and chelators play an important role in metal uptake, their transport through roots, shoot, and finally into the grains. There are two chapters that cover GE using chelators and transporter genes. Mineral bioavailability and lesser/no heavy metal is important for the success of the biofortification, and antinutrients content have to be reduced or balanced to improve the mineral bioavailability. There are chapters exclusively dealing with these aspects. With the recent advances in NBTs, intellectual property rights have gained much significance to protect their novel ideas, processes, or products, which has become a new currency to protect commercial interest in this competitive world. Authors have provided detailed information on this area. Certainly, there are many challenges associated with the successful deregulation of GE products; it has been well explained by taking an example from the Philippines regulatory system. The prospects and challenges and economic feasibility of the GE or GEd products have been elaborated. We hope that this volume will address the needs of researchers, students, agronomists, nutritionists, and law and policy makers who work or have interest in this particular field of research.

We are thankful to the all the esteemed contributors who accepted to word their research studies and opinions in this field, and to our Deputy Director General-Research, Rice Breeding Innovation Platform Lead, Rice Genetic Division and Validation Unit Head, and Healthier Rice Program Lead, IRRI, for their approval, continuous support, and encouragement to compile this book, and to all the staff of Academic Press, Elsevier, USA, Inc., for compiling this volume. We would like to thank all the healthier rice team staff of the International Rice Research Institute (IRRI) for their support.

B.P. Mallikarjun Swamy ¹ , Anca Macovei ² , and Kurniawan Trijatmiko ¹ ,      ¹ International Rice Research Institute, Philippines,      ² Department of Biology and Biotechnology, University of Pavia, Italy

Chapter 1: Molecular mechanisms leading to grain Zn accumulation in rice

C.N. Neeraja, and K. Suman     Biotechnology Division, ICAR-Indian Institute of Rice Research, Hyderabad, Telangana, India

Abstract

Deciphering molecular mechanisms vital for identifying key genes associated with high grain Zn in rice. Rice adapted complex strategies for the acquisition of Zn from the rhizosphere, its uptake by roots, its transport from root to shoot, and its movement in shoots, nodes, and leaves to the meristematic tissues and grain. Zn metabolic processes are regulated temporally and spatially by several genes. A few major gene families, viz., zinc-iron regulated proteins, heavy metal ATPases phytosiderophores like nicotinamide, and Yellow Stripe-Like transporters, natural resistance-associated macrophage proteins, and metal tolerance proteins associated with Zn uptake in rice have been characterized. Here, we review recent developments in understanding the molecular mechanism of Zn metabolism in rice and its implications in the development of Zn-biofortified rice varieties.

Keywords

Biofortification; Genes; Molecular mechanism; Rice; Transporters; Zinc

1. Introduction

Micronutrient malnutrition or hidden hunger is widely spread in low- and middle-income countries, with children and women being the most vulnerable. According to FAO estimates of 2019, around 340 million children suffered from micronutrient deficiencies across the world (FAO, 2020). Poor diet quality and excessive dependence on staple cereals are observed to be the major reasons for micronutrient malnutrition. Dietary diversification, micronutrient supplementation, food fortification, and biofortification are some of the possible key strategies to address micronutrient malnutrition (Bouis et al., 2019). Among various approaches, biofortification is one of the promising and proven strategies to alleviate micronutrient malnutrition (Bouis and Saltzman, 2017). Biofortification is the process of increasing the density of vitamins and minerals in a crop using either conventional methods through plant breeding or genetic engineering or through agronomic practices (Welch and Graham, 2004).

Rice (Oryza sativa L.) is a major staple food across the world. Biofortified rice with enhanced micronutrients could be a feasible solution for reducing the burden of micronutrient malnutrition in countries where rice is the principal component of the regular diet (Zhao et al., 2020). The availability of genetic variability for the micronutrients in grains is the primary requisite for the development of biofortified rice. Wide genetic variability exists for various micronutrients and vitamins in grains of rice (Huang et al., 2020). Brown rice (without husk and polishing) consists of endosperm (∼90% of grain weight), bran layers (∼6%–7%), and embryo (∼2%–3%). The bran layers constitute aleurone and pericarp and contain most of the grain proteins, vitamins, and micronutrients (Ram et al., 2020; Wu et al., 2016). But people generally prefer to consume polished (or milled) rice, which is mostly the endosperm after the removal of embryo and bran layers through processing. The nutritional value of polished rice is therefore determined by rice endosperm, which mainly comprises starch (70%–80%), with proteins about 7%–10% and lipids in small proportions of about, less than 1% (Yang et al., 2019). Among the minerals, Zn has a relatively stable distribution in aleurone and endosperm with a threefold difference across different rice grain fractions though ∼40% of grain Zn is lost during the polishing (Iwai et al., 2012; Lu et al., 2013). Zn content of brown rice ranged from 7.3 to 58.4 ppm and Zn content of polished rice ranged from 4.8 to 40.9 ppm with polishing losses ranging from 11.1% to 28% (Rao et al., 2020). The baseline derived from Zn content in polished rice of the popular rice varieties ranges from <12 to 14 ppm (Swamy et al., 2016). Targeting an average of approximately 30% of the estimated average daily requirements, the target Zn content was set as 28 ppm in polished rice of biofortified varieties (Trijatmiko et al., 2016). More than 10 biofortified rice varieties with a high grain Zn content of 24–27 ppm have been released in Asian countries viz., Bangladesh, India, and the Philippines through conventional breeding strategies (HarvestPlus FAO, 2019). For rice plants, Zn is a vital element needed for developing tissues such as meristems and reproductive organs, proper growth, and development of rice. Zn exists only as Zn²+ in rice and is not involved in cellular oxidoreduction reactions. Hence, Zn is an essential nutrient serving as cofactor to several enzymes involved in carbohydrate, nucleic acid, protein, and lipid metabolism at the cellular level (Ishimaru et al., 2011).

The molecular mechanisms of Zn metabolism for uptake by the roots, movement within the plant, preferential delivery to the active tissues, and grain loading are still being elucidated in rice. The understanding of Zn metabolism in rice is critical not only from the biofortification perspective but also for the Zn homeostasis mechanism. Deciphering the molecular mechanisms of metabolism leading to high grain Zn and identification of candidate genes associated with high grain Zn would accelerate the rice biofortification programs across the world. By deploying the knowledge of identified genes, superior alleles can be identified for facilitating efficient, targeted, and focused development of Zn biofortified rice varieties.

In the present chapter, the information about gene families associated with Zn metabolism was briefly outlined. The reported genes involved in the processes of Zn metabolism of rice across different tissues, viz., from root to grain have been discussed in detail.

1.1. Genes and gene families associated with Zn metabolism

Bioinformatics of candidate genes and gene families associated with Zn in other plant species like maize, Arabidopsis, or barley, initially provided the base for rice studies (Gross et al., 2003; Borrill et al., 2014). Based on publicly available rice genome sequences and gene ontology, several putative candidate genes are also being identified. Expression analysis, mutants, mapping with candidate genes-related primers, and cloning lead to the identification of a few genes associated with Zn metabolism in rice (Yamaji et al., 2013). Knowledge about genes associated with Zn homeostasis for maintenance of optimum cytosolic concentrations by compartmentalization or efflux of Zn is also essential for understanding Zn metabolism. The concentration of Zn should be maintained at an optimum level in cells so that there is no deficiency or toxicity (Bashir et al., 2013). Thus, the coordination of several genes through their temporal and spatial expression and their regulation is required for Zn metabolism in plants. The role of the genes in the processes viz., uptake, movement, transfer, accumulation, sequestration, and detoxification across the cells and tissues of rice plants needs to be further studied.

Several major gene families like zinc-iron regulated proteins (ZIP) (Krishna et al., 2020), heavy metal ATPases (HMA) (Zhiguo et al., 2018), phytosiderophores like nicotinamide (Kawakami and Bhullar, 2020), and Yellow Stripe-Like (YSL) transporters (Menna et al., 2011), natural resistance-associated macrophage proteins (NRAMP) (Curie et al., 2000), and metal tolerance proteins (MTP) (Ram et al., 2019), associated with mineral uptake have been well characterized in rice (Table 1.1). Interestingly, many of the genes or gene families are associated with the metabolism of more than one cation like Zn and Cadmium (Cd), Zn and Fe, Zn, and manganese (Mn) (Ludwig and Slamet-Lodin, 2019). In rice, some of the molecular pathways for Zn and iron (Fe) appear to be in common, viz., Iron Regulated Transporter 1 (OsIRT1) (Lee and An, 2009), Vacuolar Iron Transporter (VIT) (Zhang et al., 2012). Thus strategies for the enhancement of grain Fe content especially through transgenics methodology have also shown an increase in grain Zn content (Kawakami and Bhullar, 2018).

1.1.1. ZIP (zinc iron-regulated proteins)

ZIP genes play an important role in the uptake, transport, utilization, and storage of several metal cations in rice (Palmgren et al., 2008). There are 16 members of the ZIP family belonging to integral membrane transporters in rice. These are distributed across various cell organelles of different plant parts (Tiong et al., 2015). The expression of ZIP genes changes based on differential Zn conditions like Zn deficiency, Zn sufficiency, and excess Zn (Ramesh et al., 2003; Ishimaru et al., 2007, 2011). Transcriptional up-regulation for some of the ZIP genes under Zn deficiency/stress has been reported in rice (Lee et al., 2010a, 2010b; Tan et al., 2019). Only a few genes of the ZIP family viz., OsZIP1, OsZIP3, OsZIP7, and OsZIP9 have been functionally characterized regarding their role in Zn, Fe, Cd, and other metal transport (Liu et al., 2019; Sasaki et al., 2015; Tan et al., 2019; Gindri et al., 2020; Huang et al., 2020). Genetic and functional redundancy was observed among ZIP genes in rice. Gene knockouts of OsZIP1 or OsZIP5 showed normal growth without any phenotypic changes and overlapping spatiotemporal expression patterns of some of the OsZIP genes suggested their genetic redundancy (Lee et al., 2010a; Liu et al., 2019).

1.1.2. Iron regulated transporters (IRTs)

IRTs are members of the ZIP family transporters associated with the uptake of Fe into root cells from the rhizosphere. OsIRT1 was found to be predominantly expressed in root cells, but its overexpression was reported to enhance the Fe and Zn in roots, shoots, and seeds (Ishimaru et al., 2006). It is localized in the plasma membrane and reported to be facilitating the transport of Fe from the rhizosphere to roots. The expression of OsIRT1 is mostly regulated by Fe availability and to some extent by Zn deficiency (Ishimaru et al., 2005). The second IRT gene, OsIRT2, is reported to be located in the plasma membrane, has a similar sequence and expression to OsIRT1, also regulated by Fe deficiency, however, not associated with Zn transport as observed from heterologous expression studies (Ishimaru et al., 2006).

Table 1.1