Citation: Neeraja CN, Kulkarni KS, Madhu Babu P, Sanjeeva Rao D, Surekha K, Ravindra Babu V (2018) Transporter genes identified in landraces associated with high zinc in polished rice through panicle transcriptome for biofortification. PLoS ONE 13(2): e0192362. https://doi.org/10.1371/journal.pone.0192362
Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, CHINA
Received: June 28, 2017; Accepted: January 21, 2018; Published: February 2, 2018
Copyright: © 2018 Neeraja et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funds for this study were received from Indian Council of Agricultural Research (ICAR) to Indian Institute of Rice Research, Hyderabad under Consortia for Research Platform (CRP)-Biofortification (
www.icar.org.in/;
www.drricar.org/).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Rice (
Oryza sativa L.) is the staple food crop of 50% of the world and a major energy source especially in the developing countries. Polished rice, the most preferred form for consumption, is a poor source of micronutrients especially iron and zinc [
1–
3]. The excess dependence on polished rice in the Asian countries was reported to be responsible for malnutrition whose daily caloric intake is mainly confined to rice [
4–
6].
Polished grains of most of the rice varieties have 12–14 ppm of zinc and provide only one fifth of daily recommended zinc requirement of ~15 mg (though varies across sex and age) [
7–
9]. Zinc constitutes an important structural, enzymatic, and regulatory component in the human metabolism and its deficiency has been ranked at fifth position among the risk factors responsible for poor health. Dietary deficiency of zinc is a substantial global public health and nutritional problem with one third of the world population at risk due to low dietary intake of zinc [
10–
13]. International Zinc Association reports that 49% of world’s population is affected by zinc deficiency with maximum adverse effect on children. Zinc deficiency is most notably accountable for growth retardation, stunting, impeded intellectual development, and vulnerability to diarrhoea and pneumonia (
www.zinc.org/health/). In order to meet at least 25% of the estimated average zinc requirement for overcoming the most severe zinc deficiency, a breeding target of 28 ppm was set in polished rice based the nutrient needs, daily food intake, retention and bioavailability analyses (
www.harvestplus.org). Thus, enhancing the zinc content of polished rice has lot of potential to address wide spread zinc deficiency problem responsible for malnutrition in developing countries. Different strategies, such as biofortification, foliar or soil application of zinc fertilizers have been suggested and demonstrated to increase the zinc content of cereals [
14,
15]. Of these, biofortification, an approach of the development of micronutrient-dense staple food crops to alleviate micronutrient malnutrition is targeted, sustainable and cost effective, hence most preferred [
16]. Since 2000, several attempts are being made in rice for zinc biofortification through conventional breeding and genetic engineering approaches. To develop high zinc rice lines using conventional breeding, characterization of genetic variation in grain zinc concentration is a prerequisite. Germplasm evaluation has shown genotypic differences with several folds of zinc concentration in brown and polished rice in landraces [
17–
21].
Using the donors for high zinc content, several breeding lines with high zinc are being developed and evaluated under HarvestPlus, international and national programs (
www.harvestplus.org;
www.irri.org) [
22–
24]. As a proof of concept of combining high zinc with high yield, varieties with high zinc in polished rice developed through conventional breeding have been nationally released in Bangladesh (2013) through HarvestPlus and in India (2016) through All India Coordinated Rice Improvement Project (AICRIP) (
www.harvestplus.org;
www.irri.org;
www.drricar.org).
Rice grain zinc concentration is influenced by a large number of plant and environmental factors depending on external zinc supply and soil conditions [
25,
26]. Several plant factors affect the uptake of zinc from soil to roots, transport to leaves and stem and remobilization to developing grains [
26,
27], thus understanding their genetics and molecular mechanism would help in identifying the critical candidate genes and their deployment in the development of high zinc rice varieties [
28]. Several genes/gene families involved in zinc homeostasis
viz., zinc related transporter (
ZRT) and iron related transporter (
IRT) like proteins comprising
ZRT/
IRTprotein family (
ZIP), heavy metal ATPases (
HMA), Yellow stripe like (
YSL), natural resistance associated macrophage protein (
NRAMP), metal tolerant protein (
MTP), nicotianamine synthase (
NAS), nicotianamine aminotransferase (
NAAT), zinc-induced facilitator-like (
ZIFL) and others have been characterized in rice. Several transcription factors (TF) like
OsNAC,
OsIDEF and
OsIRO also showed to play an important role in up regulating the genes involved in metal homeostasis [
29]. Through expression analyses, several genes were shown to be associated with enhanced iron and zinc concentrations in different tissues in rice [
30–
34], however, the role of different families of genes in zinc metabolism of rice yet to be elucidated [
35]. It appears that the expression of genes associated with zinc metabolism varies under different situations of zinc availability either deficient or sufficient or excess in soil [
36–
42]. Zinc localized in the seed aleurone layer chelates with phytic acid, main form of inorganic phosphate (Pi) storage in seeds to form phytate, a salt of inositol phosphate and inhibits zinc solubility, digestibility and absorption in human body [
43–
45].
Though several genes associated with zinc metabolism have been characterized in rice, very little is known about how zinc is transported from leaf xylem to phloem of developing seeds and ultimately unloaded into seeds [
3]. Physiological and transcriptome analyses of response to zinc deficiency of two rice lines with contrasting tolerance was reported to be determined by root growth, maintenance and organic acid exudation rates, and not by zinc-transporter activity [
46], but microarray analysis of zinc deficient rice showed up regulation of several genes involved in zinc transport [
39].
With the advent of time, RNA-Seq has become the most important approach to study gene expression profiling using the next generation sequencing technologies providing a more precise measurement of gene transcripts dynamics on global scale in different tissues and biological contexts [
47]. Rice landraces are known source of many desirable traits and have been characterized with high zinc in polished rice, although low yielding as opposed to improved varieties [
19,
20]. In general, to find out the genes associated with traits of interest in rice landraces, the transcriptome studies comprise differential treatments of susceptible and tolerant genotypes (mostly landraces) under stressed conditions
viz., drought, salt and cold, deficient or excess nutrients versus control conditions for the differential expression of genes [
48–
53]. With an objective of identification of the genes exclusively associated with high zinc in polished rice, those would be efficient in regular rice growing conditions as prevail in the farmers’ fields, the present study was conducted under regular or zinc sufficient situation. To identify the genes responsible for higher zinc in polished rice, RNA-Seq based transcriptomic analyses of developing panicle of two landraces and a widely grown popular variety with differential zinc in the polished rice were compared and a set of 311 up regulated genes and 534 down regulated genes in two landraces were identified and the six promising genes were validated through quantitative real time PCR (qRT-PCR) and their association with zinc in polished rice through mapping.
Discussion
Biofortification of rice for high zinc in rice appears to be promising strategy for addressing the some of the malnutrition issues in developing countries, especially for those whose major diet is polished rice with poor micronutrients. The development of varieties with high zinc would be relevant to alleviate malnutrition, but the lack of information on translocation of nutrients from vegetative tissues to grains is one of the barriers to rice biofortification [
40,
66,
67]. Several donors for high zinc in polished rice have been identified through the evaluation of landraces and are being used in development of high zinc breeding lines [
18–
21,
68]. In parallel, several studies are being conducted on mechanism of zinc uptake and its translocation into the grain [
41,
42,
69–
73]. However, the information on genes associated with zinc uptake and its translocation are very limited in rice, but for reports on zinc transporters and ZIP genes [
27,
74–
76]. Some transgenics of rice with metal chelating molecules like nicotianamine, IRT, 2’-deoxymugineic acid (DMA) targeted for the enhanced iron content also showed increased zinc content in grain, thus role of a few candidate genes in zinc homeostasis is available [
27,
77–
83]. Since, physiological studies of zinc in rice have shown transfer of zinc from the vegetative tissues to reproductive tissues to be constraint for achieving high zinc in rice grain, we attempted to characterize a set of genes expressed in developing panicles of two landraces (CTM and KJJ) with high zinc in the polished rice in comparison with a popular improved high yielding rice variety with low zinc content in polished rice (BPT) grown under sufficient zinc soil conditions. Most of the work on zinc nutrition in plants has concentrated on genes and pathways related to extreme phenotypes, such as zinc deficiency and zinc excess-derived changes in growth and/or bulk concentrations in shoots or roots [
35,
39,
84]. In general, to identify the differential expressed genes associated with nutrients, excess or deficient conditions are studied along with control [
46,
48,
50]. However, to identify the set of genes responsible for high zinc under general irrigated rice cultivation conditions as practiced by the farmers with fertilization of zinc or native soil zinc, the genotypes were grown under regular soil with sufficient zinc. The two rice landraces of the present study appeared to be promising donors for the high zinc content in polished rice reiterating the fact that the landraces to be the source of novel genes/alleles for traits of interest as observed for stress tolerance and other traits in rice [
85,
86].
Out of the three genotypes, zinc in straw content was ~18 to 30% more in BPT than CTM and KJJ, however, the zinc content in grain was ~ 42 to 45% more in brown rice and 30 to 35% in polished rice of CTM and KJJ suggesting the possibility of efficient translocation of zinc into grains by landraces than BPT. Similar trend of differential translocation of zinc has been reported by Johnson-Beebout et al. in two genotypes
viz., in IR68144, a substantial amount of zinc stored in the stem has not translocated into grain, whereas in another genotype, IR69428 has more zinc in the grain even with lower concentration of zinc in the stem[
73]. Wide genotypic variation of zinc uptake, its content in stems, leaves, panicles and grains has been reported in rice [
41,
71]. Wissuwa et al. concluded that grain zinc concentration is largely determined by genotype rather than by zinc fertilization, which could be attributed to differences in zinc uptake behavior [
26]. The mechanism of translocation of zinc into grains also appears to be different based on the zinc availability (sufficiency versus deficiency) [
36,
37,
40,
69,
73]. The studies on zinc concentrations of panicle and stem between flowering and grain maturity stages suggested a relative transfer barrier between the vegetative and reproductive tissues, but for the same set of genotypes the zinc concentrations of grain were higher than the concentrations of the panicle implying that the loading of the grain from the panicle to grain is easier than loading of the panicle from the stem and sheath [
41]. Thus, in the present study we could identify two landraces with efficient mechanism of translocation of zinc from stem into the reproductive tissue.
An interesting phenomenon called dilution, in case of decrease of nutrient concentrations in plant tissues with the dry matter increase is generally observed in cereals [
87], thus explaining the inverse relationship of zinc content and yield [
88]. In the present study, in the mapping population of BPT and CTM, we could identify ~10 promising lines with desirable recombinants of high yield and zinc content (>28 ppm).
To identify the genes associated with differential zinc content of polished rice in the panicles among the three genotypes, the whole transcriptome of three rice cultivars was analyzed and a large number of differentially expressed genes along with novel transcripts associated with trait of interest were identified. The RNA-Seq of BPT, CTM and KJJ of developing panicle before booting stage resulted in 106296448 high quality reads with 82–86% of alignment attributed to mapping of
indica genome using
japonica reference genome (
Table 2). Mapping of reads through the transcriptome studies of
indica or
japonica or wild species of rice to the reference genome Nipponbare ranged from 69% to 98% based on the subspecies, stage and tissue of the genotypes [
51,
52,
89]. The overall transcripts are more in BPT, an improved variety; however the exclusive transcripts are more in the landraces, thus proving that the native germplasm to be the resource of novel alleles/genes for several traits of interest in rice. The pair wise comparison of genotypes for common and exclusive transcripts also confirmed the abundance of exclusive transcripts in landraces.
More number of transcripts was up regulated in BPT as compared to both landraces and the number of the down regulated genes appears to be more in the landraces supporting the similar observations in landraces N22 and Pokkali under control conditions [
89]. The conscious selection for more yield and other favourable agro-morphological traits during the development of improved varieties could have played role in the pooling of many up regulated genes for their expression in terms of phenotype. Only a small fraction of transcripts was found to be differentially regulated in this study from the analyses of the data confirming the earlier reports that at a particular stage for particular tissue, only a fewer number of stage and tissue specific differential transcripts are observed in rice [
90].
Differential expression of transcripts was observed for all the three genotypes of the study. Among the three, there were only 37 common transcripts for pair wise comparisons with 563 common transcripts for genotype wise comparisons. Most of the differentially expressed genes in landraces are uncharacterized proteins, suggesting the existence of novel genes/alleles in the landraces.
Interestingly, not many obvious candidate genes associated with zinc metabolism found to be up or down regulated in the present study. Similar observations were also reported by Astudillo-Reyes et al. in their transcriptome study of developing pod of two common bean genotypes with contrasting zinc concentration grown under regular (zinc sufficient) conditions [
91]. The information about
OsZIP genes during different stages of flowering and seed development reported to be scarce but for
ZIP genes in anthers.
Insilico analyses has shown enhanced temporal expression of
OsNAS1,
OsNAS3,
OsNAAT1, 2’-deoxymugineic acid synthase 1 (
OsDMAS) during the flowering and seed development of Nipponbare, reference genotype of rice [
27]. However, in our transcriptome study of three genotypes, differential expression of these genes was not observed in the panicle tissue at that time point of sample collection. Little genotypic variation in transcript abundance of zinc responsive root zinc transporters, P-type ATPases,
HMA,
OsYSL,
MTP1 and
MTP3 was observed between the RIL46 (a zinc deficiency tolerant line) and IR74 (a zinc deficiency sensitive line) [
46]. However, microarray analysis of zinc deficient rice with root and shoot tissues revealed the up regulation of several genes involved in zinc transport [
39]. The threshold levels of detection of differential expression needs to be compared between RNA-Seq and microarray for the zinc metabolism genes. Studies showed differential expression of candidate genes associated with zinc metabolism in flag leaves in genotypes with differential zinc, but expression studies of the association of candidate genes of zinc metabolism in panicle are few in rice [
31,
92].
Two known genes with their association with zinc and iron metabolism
viz.,
NRAMP5 and Vacuolar Iron Transporter (
VIT) also showed up regulation only in CTM and KJJ, which were further validated by qRT-PCR analyses. The
NRAMP family of transporters appears to regulate nutrient export from the vacuole [
93]. The role of rice
NRAMP5 has been characterized in manganese, iron and cadmium transport in root tissues [
94,
95] and differential expression of
NRAMP5 in root tissues of genotypes with differential iron and zinc was also reported [
32]. In the context of high zinc content in rice grain,
NRAMP5 may play a critical role in zinc homeostasis/mobilization of zinc from panicle to grain. Transcript for
VIT homolog 5 involved in showed significant up regulation in landraces. Vacuolar sequestration is another mechanism to enhance the concentrations of iron and zinc in seeds [
96]. Transporters belonging to several different families transport metals between the cytoplasm and the vacuole including the vacuolar membrane transporters
viz.,
OsVIT1 and
OsVIT2 to modulate Zn
2+ and Fe
2+ import to the vacuole and translocation between flag leaves and seeds in rice. Disruption of the rice
VITorthologues (
OsVIT1 and
OsVIT2) increases iron and zinc accumulation in rice seeds and decreases iron and zinc in the source organ flag leaves, probably because VIT genes are highly expressed in rice flag leaves [
97–
99]. Thus, it can be hypothesized that the activity of
VIT5 could contribute to high zinc in polished rice.
The interesting observation of four proton-coupled peptide transporters (
POT) family proteins with differential expression exclusively in landraces suggests for their role of possible nutrient metabolism. The POT/PTR family proteins are mainly involved in cellular uptake of small peptides and route the uptake of amino acids and nitrogen. The up regulation of peptide transporters corroborated well with the higher fold expression in CTM and KJJ as compared to BPT through qRT-PCR. In the phloem, zinc is thought to be transported either as Zn–NA or complexed with small proteins [
42,
100]. Proteins that transport micronutrient–NA complexes have been identified recently as YSL proteins, which are members of the oligopeptide transport (OPT) family [
100–
102]. A proton-coupled symporter ZMYS1, was shown to function for the uptake of phytosiderophore and nicotianamine–chelated metals in maize [
103]. The role of POT family in the nutrient metabolism though reported, further characterization and cloning of these genes is needed for confirming their exact mechanism of action [
104]. Higher expression of the PHO exporters
viz.,
PHO 1–3 in landraces may suggest their possible function for the micronutrient concentration in the panicle tissue. The involvement of OSPHO1; 1 in the regulation of iron transport through integration of phosphate and zinc deficiency signaling in rice has been already reported [
105]. Out of three rice
PHO1 genes identified, only
OsPHO1;2 was shown to play a key role in the transfer of Pi from roots to shoots and regulated by Pi deficiency, while
OsPHO1;1, and
OsPHO1;3, are still to be characterized [
106]. Among other transporter genes potentially linked to altered zinc nutrition, nine putative phosphate transporters showed increased root expression in zinc deficiency tolerant rice line under zinc deficiency [
46]. The role of
PHO transporters in zinc metabolism as observed in this study is to be elucidated in detail,
PHO genes could play role in tripartite nutrient PiZnFe interaction in plants [
105,
107].
Out of the five transcripts for potassium channel and transporters,
AKT2 was up regulated in CTM and KJJ than BPT, but the
KOR 1 and
KOR 2 transcripts functioning as voltage-gated potassium channel were up regulated in BPT than the landraces. Since, zinc is an essential micronutrient for plant; its uptake is needed for general plant metabolism as evident from the higher concentrations of zinc in the vegetative parts in BPT, an improved variety (
Table 1). However, our interest is of the genes associated with high zinc in polished rice playing role in the translocation of zinc to the reproductive tissue. Though the potassium transporters are characterized, their role in micronutrient uptake and mobilization is yet to be explored [
108]. In our study, the higher expression suggests its potential for characterization for involvement in micronutrient or zinc content of plant metabolism.
Transport proteins embedded within membranes are key targets for improving the efficiency with which plants take up and use water and nutrients [
109]. Various transcripts for
ABC,
NRAMP, phosphate, potassium, peptide, vacuolar iron transporters showed differential expression in our data and we suggest their possible role for the high zinc content in polished rice (
Table 3). The transporters which are comparatively up regulated can be assumed to be actively involved in metabolic processes at panicle initiation stage. The uptake of mineral elements is mediated by various transporters belonging to different transporter families. Thus, the plant transporters can be effectively deployed for improving the uptake of nutrients and water as to enhance the yield and micronutrient in the grains [
74].
Differential expression of Os03g0839200-
MATE (multidrug and toxic compound extrusion) efflux family protein was also observed in both landraces. MATE effluxer genes were overrepresented among genes that were differentially expressed in roots between two rice genotypes with differential response to zinc deficiency under different zinc conditions and were hypothesized to be possible candidates for organic acid (OA)/DMA efflux transporters [
46]. Natural variation at the
FRD3 MATE transporter locus revealed cross-talk between iron homeostasis and zinc tolerance in
Arabidopsis [
110]. Several other genes
viz., kinases, peptide transport protein, homeobox-leucine zipper protein, transcription factor were also up regulated in landraces and are under being validation.
Among the transcription factors, OS02G0810900 Putative NAC domain protein NAC1 showed significant log fold change and confirmed by qRT-PCR analyses in the present study. Differential transcripts for zinc finger (
ZF) (five),
WRKY (four),
MYB (two),
AP2,
bHLH,
EREB,
ZIM and heat responsive TFs were also found. The transcripts for
ZF TF,
ZFP30,
WRKY 5 and
ZIM motif TF showed exclusive expression only in the landraces. Transcription factors are integral in linking sensory pathways to many responses. Core sets of transcription factor family genes are differentially expressed in earlier studies including basic leucine zipper (
bZIP),
WRKY,
MYB, basic helix-loop-helix (
bHLH), and NAC families have been reported in nutrient homeostasis studies in rice. These transcription factors, in turn, regulate the expression levels of various genes that may ultimately influence the nutrient content in rice [
111]. Nishiyama et al. reported that metal-chelate complexes are formed in rice phloem sap and this transport is critical for grain zinc content. The observed enhanced expression of one
NAS3 transcript (OS07G0689600) in the present study could be playing an important role in the accumulation of zinc in rice grain [
112]. One of the important gene families associated with nutrient remobilization from source organs to developing seeds is the
NAC (
NAM,
ATAF, and
CUC) family of TFs [
113]. Enhanced expression of
OsNAC5 expression in flag leaves and panicles and its association with higher seed iron and zinc concentrations was reported earlier in rice [
31,
114]. Ricachenevsky et al. discussed in detail about the role of
NAC factors in relation to leaf senescence with iron and content in the seed [
115]. The validation of the
NAC 1 gene for its association in zinc content in polished seeds is under progress. Among the zinc finger TFs, the expression of
ZF1 was higher in KJJ than CTM and BPT predicting its role during panicle development. Though the role of zinc- finger transcription factors in the important biological processes of plants has been studied, their involvement in zinc metabolism in grains is yet to be confirmed [
116,
117]. The role of
WRKY factors is nutrient metabolism is being explored recently. The
WRKY 74 has been shown to play regulatory role in phosphate uptake and mobilization [
118,
119]. On similar lines, it can be proposed that
WRKY5 and other
WRKYfactors could be involved in regulating the zinc metabolism/ translocation in rice. Two members of the basic region/leucine zipper motif (
bZIP) transcription factor gene family,
bZIP19 and
bZIP23, were shown to coordinate the adaptation of
Arabidopsis to low zinc phytoavailability [
120]. The
bHLH TF has been reported to regulate OsIRO2 play an important role in iron homeostasis [
121]. In this study also, the expression of bHLH is evident of regulating the genes involved in zinc homeostasis but it would need further characterization. The TF APG found to be highly expressed in KJJ is a typical bHLH transcription factor that acts as negative regulator of grain size (grain length and weight by controlling cell elongation in lemma and palea) [
122]. The role of these TFs till now has been characterized to some extent in rice roots, but their involvement during panicle initiation or grain filling and their association with nutrient uptake is yet to be elucidated. The MYB TF has been reported in the interconnection between zinc and inorganic phosphate homeostasis in
Arabidopsis, namely the
MYB transcription factor
PHR1, the Pi exporter
PHO1 [
123]. In our study also,
PHO transporters and its homologues are highly expressed. Hence, it can be put forward that this
MYB TF highly expressed in KJJ can be a candidate gene for zinc mobilization from the panicle to the rice grain. The controlled and regulated uptake and mobilization of micronutrients is very essential for maintaining homeostasis and ionic concentration in the cell. High concentration of metal may lead to toxicity and disturbances in its cellular function, thus the metal-responsive transcription factors have been reported to regulate trace metal metabolism. Moreover, TFs have been shown to play a critical role in the regulation of the levels of protein, zinc and iron in the mature grain [
124]. In this study, genes from eight TF families were identified to be associated with the trait of interest, whose validation is in progress.
Some of the genes with significant fold change as indicated viz., peroxidase, nodulin-like protein and others can also be explored for their function in the zinc metabolism. Characterization of differentially expressed uncharacterized transcripts is also being targeted as they may play an important role in accumulation of zinc in polished rice. Through GO enrichment analysis, we conclude that out of 12 pathways, the amino acid metabolism, biosynthesis of other secondary metabolites, carbohydrate metabolism, folding, sorting and degradation pathways may contribute significantly to enhanced zinc content in polished rice.
The co-localization of 24 differentially expressed transcripts for
MYB,
bHLH, serine threonine kinases, RING zinc finger proteins with mQTL reported for zinc, zinc and phytate corroborated with the up regulated transcripts for phosphate transporters/ translocators in the landraces [
59].
In order to validate the information of the differentially expressed genes generated in this study in deployment in marker assisted selection (MAS) for high zinc in polished rice, candidate gene based markers were developed for a differentially expressed gene,
NRAMP5 as a proof of concept. Though polymorphism was observed, the resolution of candidate gene based markers into parental alleles in the RIL population of the study was poor on agarose gel and the sequence information of the polymorphic product could not generate efficient marker system. Thus, RM markers spanning six differentially expressed genes confirmed with qRT- PCR were selected for their validation for association with traits of interest. And out of six genes, five differentially expressed genes showed association with zinc content in brown and polished rice, thus validating the differential expressed genes and their association (
Table 4). Thus, we have shown the deployment of transcriptome data for the generation of the differentially expressed genes from the novel germplasm sources and their utility as markers system for MAS in our study.
The reported association of zinc and iron content in the grain suggests some level of common regulatory mechanisms for their metabolism [
45], so analyses of both elements was done for plant samples (including straw and grain) and mapping population (grain). However, iron content in polished rice is much below the target iron content set by Harvestplus, thus, only limited analyses was done for iron in the present study (
S5 Table).
The development of zinc biofortified rice is challenging due to the complexity of genetic and metabolic networks controlling the homeostasis of zinc [
38,
111,
125]. Genotypic variability needs to be characterized for the uptake, remobilization and concentration of zinc in polished rice, which are affected by use efficiency of zinc source-sink relations [
40]. Several transporter gene families appear to be associated with high zinc metabolism in polished rice. Thus, in present study, we have identified two landraces with promising zinc content in polished rice and we have validated the differential expressed genes identified through transcriptomic studies in the mapping population. Further studies are needed to target earlier and later developmental time points along with different tissue samples to better characterize genotypic differences in zinc remobilization with focus on functional characterization of zinc transporters
in planta, elucidation of zinc uptake and sensing mechanisms, and on understanding the cross-talk between zinc homeostasis and other physiological processes [
35].
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