ZIP genes are involved in the retransfer of zinc ions during the senescence of zinc-deficient rice leaves

doi:10.21203/rs.3.rs-3110004/v1

Download PDF

Research Article

ZIP genes are involved in the retransfer of zinc ions during the senescence of zinc-deficient rice leaves

https://doi.org/10.21203/rs.3.rs-3110004/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Zinc (Zn) is essential for human health, but rice often lacks sufficient amounts. Leaf senescence is a vital developmental stage during which nutrients are redistributed to other organs. However, Zn retransfer during deficiency is often overlooked. In this hydroponic experiment, we studied the effect of Zn deficiency on rice seedlings, specifically focusing on the fourth leaf under control and deficient conditions. Growth phenotype analysis showed that the growth of rice nodal roots was inhibited under Zn deficiency, and the fourth leaf exhibited accelerated senescence and increased Zn ion transfer. We conducted transcriptome analysis on the fourth leaf at two time points. The fourth leaf at the second time point under control conditions was used as the control and compared to when Zn ion content markedly decreased under control and deficiency conditions. Analyzing differentially expressed genes showed that Zn deficiency regulates more ZIP family genes involved in Zn ion retransfer in the leaves. OsZIP3 upregulation under Zn-deficient conditions may not be induced by Zn deficiency, whereas OsZIP4 is only induced during Zn deficiency. Gene Ontology enrichment analysis showed that Zn-deficient leaves mobilized more biological pathways (BPs) during aging, and the enrichment function differed from that of normal aging leaves. The most apparent “zinc ion transport” BP was stronger than that of normal senescence, possibly due to Zn-deficient leaves mobilizing large amounts of BP related to lipid metabolism during senescence. These results provide a basis for further functional analyses of genes and the study of trace element transfer during rice leaf senescence.

zinc deficiency

rice

ZIP

zinc ion retransfer

leaf senescence

Zinc (Zn) is an essential trace element for plant growth and development. However, Zn deficiency is widespread in crops worldwide, making it the most common micronutrient deficiency [1, 2]. In addition, because Zn from the edible parts of crops is the primary source of Zn for humans, Zn deficiency in crops can lead to Zn deficiency in humans [3, 4]. Therefore, understanding the absorption and distribution of Zn in rice is essential.

Zn plays a vital role in promoting the growth and development of rice and ensuring high yield and quality. However, owing to the rich and complex soil resources available for rice cultivation worldwide, the distribution of Zn content in different soils varies greatly, and the ability of Zn uptake and accumulation in rice with different genotypes also varies [5, 6]. This results in poor Zn uptake and enrichment in rice. Rice primarily relies on its roots to absorb Zn from the soil and Zn fertilizer [7, 8]. The effectiveness of metal extraction from the soil is controlled by three conditions [1, 2, 9]: (1) soil conditions (upland or flooded soil, pH value of soil solution); (2) mineralization (ionization and hybrid formation); and (3) transporter uptake. Root absorption can also be divided into three steps [10]: (1) rapid Zn adsorption on the surface of root cells, which does not require energy consumption; (2) entry inside the root through the apoplast or symplast pathways; and (3) entry of the duct or tracheid through passive and active transport from the cell membrane into the root cell. In the root, Zn²⁺ is loaded from the xylem parenchyma cells into the xylem [11].

After the roots absorb Zn, it is transferred to parts with high Zn demand [12]. Owing to the high demand for Zn for building and operating the photosynthetic apparatus of chloroplasts in photosynthetic plant tissues [13], the greatest proportion of Zn is allocated to leaves. During leaf development, photosynthetic organ biosynthesis is dominant, and the Zn absorbed by the roots is primarily distributed in the leaves. However, leaves age and become pools of organic and inorganic matter [14]. During leaf aging, Zn is released due to organelle degradations and then migrates to new tissues through the phloem [15, 16]. This process is known as Zn retransfer. Zn is highly mobile in plants and can be redistributed from old to new leaves, ears, and grains through the phloem, especially during Zn deficiency. The retransfer of Zn stored in leaves greatly contributes to the mineral content of rice grains [17].

Zn accumulation in grains depends on the Zn absorbed by roots from the soil and Zn transfer accumulation during leaf aging (especially in Zn deficiency). Therefore, understanding how Zn transfer in aged leaves can be improved is essential. In this study, we used the fourth leaf of rice seedlings as the research object to determine the time point of Zn retransfer during senescence through ion analysis and explored the critical factors of Zn retransfer during leaf senescence. In addition, we compared the growth phenotypes of Zn-deficient and control plants.

Plant material and growth conditions

The rice variety Zhonghua 11was used in this experiment. The seeds were disinfected with 50% sodium hypochlorite solution for 45 min. After five washings with deionized water, seeds were immersed in deionized water in the dark for 2 days at 38℃. Subsequently, the seeds germinated in the dark at 25℃ on wet filter paper for three days. After germination, the seedlings were transplanted into a hydroponic box containing deionized water for two weeks. Deionized water was replaced with the IRRI (International Rice Research Institute) formula nutrient solution, and the culture was continued under control or treatment conditions. Two static conditions were used: control (0.75 µM Zn) and Zn deficiency (0 µM Zn). The first sampling time was set after the fourth leaf was fully developed, and samples were taken four times every 7 days. The fourth leaf and the root systems were sampled. The samples were collected for (i) root and leaf phenotypic analyses, (ii) ion analysis, and (iii) RNA sequencing. The nutrient solution was replaced every 3 days, and the pH was adjusted to approximately 5.4. Harvesting took place 2 h after the beginning of the afternoon. The growth conditions were 14 h of light, 28℃; 10 h of darkness, 20℃. During all experiments and conditions, the hydroponic boxes and solution containers were washed before using 6N hydrochloric acid to remove Zn traces.

Ion analysis

After harvesting, the fourth leaves were dried at 65℃ for 4 days and then digested with nitric acid [18]. Inductively coupled plasma mass spectrometry was used to measure the Zn concentration.

RNA sequencing

After sampling, the tissues were frozen in liquid nitrogen and stored at -80℃. Nine samples were used (tissue: a fourth leaf; the second time point of the control group was used as the control and compared with the fourth time point of the control and Zn-deficient groups with three replicates). Total RNA was extracted, and the RNA quality was tested. Only RNA of good quality was constructed for subsequent libraries. The detection of RNA samples primarily included the following methods: (1) agarose gel electrophoresis to analyze RNA degradation and whether there was contamination; and (2) RNA purity was measured using a Nanodrop (OD260/280 ratio). After the samples were qualified, oligo (dT) beads separated mRNA. The mRNA was divided into short fragments by adding an interrupting reagent, which was used as a template to synthesize the first cDNA strand, and Second Strand Synthesis Mix was added to synthesize the second strand. After terminal complementation and adding “A,” The RNA-Seq adapter was connected, and the connected products were purified for PCR amplification. The 300–600 bp target products were purified using magnetic beads for sequencing. After library construction was completed, a Qubit was used for preliminary quantification. The library was then diluted to 1 ng/ul, and a Qseq100 DNA Analyzer was used to detect the insert size of the library. Quantitative PCR was used to accurately quantify the effective concentration of the library (effective concentration > 2 nM) to ensure its quality. After library inspection, different libraries were pooled according to the requirements of effective concentration and the target amount of data, and the libraries were sequenced on the Illumina NovaSeq 6000 platform using the PE150 sequencing strategy. Raw data were filtered using fastp software, which includes the following steps: (1) primers and adaptor sequences were removed; (2) removal of sequences with fragment lengths < 50 bp; (3) removal of the N base that reached a certain proportion of reads (default: 5 bp); (4) low-quality bases with mass values < 20 were removed; and 5) four bases were used as windows to calculate the average base mass. The base was removed if the average base mass was < 20 (Q20). Hisat2 was used to map the similarity between clean data and reference genomes. The overall mapping rate of the nine groups was > 96%.

Data analysis

edgeR was used for differential expression gene (DEG) analysis. The DEGs in the standard were as follows: Calculated expression ratio change (fold change) met | log2 (fold change) | > 1; 2. The Q value was obtained by FDR correction of the P value, and both P value ≤ 0.05 and Q (FDR) value ≤ 0.05 were satisfied. Genes above the criteria were considered differentially expressed and used for principal component analysis (PCA). The Gene Ontology (GO) database was constructed using XXXggnog, and clusterProfiler in R was used for GO enrichment analysis of differential genes with an adjusted P value of ≤ 0.05. Other statistical tests were conducted using analysis of variance or Student’s t-test.

Zn deficiency inhibits rice seedling shoot growth and development

Zn deficiency negatively affected rice seedling shoots. The plants were slightly yellowish (Fig. 1), and the shoot height was shorter than that of the control. With increased treatment time, shoot inhibition in the Zn-deficient treatment group increased, and the difference in shoot fresh weight between the Zn-deficient treatment and the control groups gradually increased. At the fourth sampling time (21 d), the shoot fresh weight of the Zn-deficient treatment group decreased by 61.29% (Fig. 2). Although the control was slightly higher than the Zn deficiency treatment at the first two time points, no significant difference in the aboveground fresh weight between the two treatments was observed. In addition, the difference between the fresh weights of the two treatment groups at the second time point was smaller than that at the first time point.

Zn deficiency affects the growth of the fourth rice leaf

Zn deficiency reduced the total biomass and dry weight of the fourth leaf, accelerated leaf senescence, and shortened the leaf growth cycle (Fig. 3). At the second time point, the fresh and dry weights of the control group were significantly higher than those of the Zn-deficient treatment group, by 19.68% and 23.35%, respectively (Fig. 3). On day 21, the fresh and dry weight of the fourth leaf of the control group was reduced by 16.42% and 17.98%, respectively, compared with that on day 7. Furthermore, the reduction in the Zn deficiency treatment group was greater, 3.39 times and 3.37 times that of the control (Fig. 3).

At the first two time points, the biomass of the fourth leaf in both treatments increased, indicating that although the fourth leaf was fully developed at the first sampling, it did not reach full maturity yet, but was fully mature seven days later. At the first time point, the fresh and dry weights of the fourth leaf of the control treatment were slightly higher than those of the Zn deficiency treatment group. However, no significant difference between the two treatments was observed, and the difference gradually increased at the second time point and later on.

Zn deficiency alters the root architecture of rice seedlings

The effect of Zn deficiency on the root growth of rice seedlings differed from that on the shoot responses. Zn deficiency inhibited root elongation and development (Fig. 1). The inhibition of fresh and dry root weights of Zn-deficient seedlings increased gradually with the extension of treatment time, decreasing by 55.92% and 47.78%, respectively, compared with the control at 21 days (Figs. 4a and b). The inhibition of total root length was similar to that of fresh and dry weight, with a reduction of 15.68% (Fig. 4c).

The difference in total root length primarily came from the roots of nodes; compared with the control, the length of node roots of Zn-deficient plants decreased by 14.53% at 21 days, whereas the seed root length was similar (Fig. 4d). The number of nodes in the Zn-deficient seedlings stopped the growth of new nodes with the extension of treatment time, and five fewer nodes were grown compared to the control plants (Fig. 4e). The biomass and length of the seed roots decreased with increasing Zn concentration.

Zn deficiency affects Zn ion transfer during senescence in the fourth rice seedling leaf

The Zn concentration and content in the fourth leaf of Zn-deficient rice seedlings were significantly lower than those of the control. From day 0 to 7, the Zn ion concentration in the fourth leaf of rice seedlings under the Zn deficiency treatment decreased significantly (Fig. 5a) and then increased slightly but did not reach a significant level (Fig. 5b). The Zn ion concentration and content in the control treatment significantly increased. At day 7 to 21, the Zn ion concentration in the fourth leaf of rice seedlings under Zn-deficient conditions showed no significant change, but the Zn ion content significantly decreased by 42.2% on day 21 compared to on day 7. The Zn ion content in the control group also decreased by 14% (Fig. 5b).

Transcriptome response to Zn deficiency

Principal component analysis of the RNA-Seq data showed that the variance in gene expression between biological replicates was very low. The three sample groups were grouped separately (Fig. 6a). PCA separation between the samples was clear along PC1. The fourth leaf sample at the second time point of the control group is located on the left side of the x-axis, whereas the fourth time point of the control and Zn-deficient groups is located on the right side of the x-axis (Fig. 6a), indicating that aging was the main component.

DEGs were identified (adjusted P < 0.05, log2 [fold change] > 1). Among the samples at the four time points in the two treatments, those at three time points were selected for comparison. The three comparisons included comparing the second point in time of the control group (C2) with the fourth point during control group (C4), and the fourth point during Zn deficiency (D4) (two comparisons, C2 vs. C4, and C2 vs. D4), and the fourth point during Zn deficiency (D4) with the fourth point in the control group (C4)(D4 vs. C4). A total of 7622 unique DEGs were identified in the three comparison sets o. In total, 828 genes were common among the three comparisons (Fig. 6c). C2 and D4 mobilized the most DEGs, whereas C4 and D4 mobilized the least (Figs. 6b and c). C2 vs. C4 and C2 vs. D4 had a high DEG overlap (Fig. 6c).

Biological pathway involvement in Zn deficiency treatment

The upregulated and downregulated DEGs were subjected to GO enrichment analyses. Some biological processes were overexpressed in all three sets of comparisons (Fig. 7). In the fourth leaf of normally aged rice (C2 vs. C4), the expression of biological pathways (BPs) related to RNA metabolism in the DEG was strongly overexpressed in the upregulated genes. Overenrichment of BP in response to stimuli, photosynthesis, and light was observed and was driven by downregulated genes (Fig. 7a). C2 in the control group and D4 in the Zn-deficient group mobilized more BPs. BP enrichment driven by the upregulated genes was primarily related to oxidoreductase activity, metabolism, stress, and metal transport reactions. The BPs were driven by downregulated genes mainly involved in cell wall synthesis, light response, and photosynthesis (Fig. 7b). Moreover, compared with C4, many D4-enriched BPs were related to stimulus-response, lipid metabolism, and Zn ion transport, all driven by upregulated genes. BPs driven by the downregulated genes related to photosynthesis and cell wall biosynthesis were also abundant (Fig. 7c). The most striking observation was the enrichment of the “zinc ion transport” BP, which was higher in C2 and D4 than in C2 and C4. This finding may be because C2 and D4 mobilized many BPs related to lipid metabolism (Fig. 7).

Zn ion transport genes are regulated differently under different conditions

Among the 7622 DEGs (Fig. 6), 11 were associated with Zn ion transport. Among the 11 genes, eight were differentially expressed in the comparison between C2 and C4, and these eight genes were also differentially expressed in the other two comparisons. One gene was differentially expressed only in the comparison between C2 and D4, and two genes were differentially expressed only in the comparison between C2 and D4, and C4 and D4 (Table 1). These 11 genes belonged to the metal transporter family (ZIP [seven genes] and MT [four genes]).

Table 1

Zinc ion transport-related genes in differentially expressed genes (DEGs)
Rice

Gene ID	Gene description	Gene name	Inclusion contrast
Os12g38300	Metallothionein-like protein 4C	OsMT1c	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os05g39560	Zinc transporter 5-like	OsZIP5	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os12g0571000	Metallothionein-like protein 4B	─	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os05g39540	Zinc transporter 9-like	OsZIP9	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os12g38051	Metallothionein-like protein 4B	OsMT-I-4b	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os12g0567800	Metallothionein-like protein 4C	─	C2_vs_C4; C2_vs_D4; C4_vs_D4
Os01g74110	Zinc transporter 1-like	OsZIP1	C2_vs_C4; C2_vs_D4
Os04g52310	Zinc transporter 3-like	OsZIP3	C2_vs_C4; C2_vs_D4
Os08g10630	Zinc transporter 4-like	OsZIP4	C2_vs_D4; C4_vs_D4
Os06g37010	Zinc transporter 10	OsZIP10	C2_vs_D4; C4_vs_D4
Os05g10940	Zinc transporter 7-like	OsZIP7	C2_vs_D4

The negative inhibitory effect caused by Zn deficiency gradually strengthened in the shoot, and shoot biomass accumulation in Zn-deficient rice became slower. In addition, Zn deficiency reduced the fresh and dry weights of the fourth leaf, accelerated senescence, and shortened the growth cycle of the fourth leaf. Notably, there was no difference in the fresh weight of the aboveground parts between the two treatments at day 0 to 7, and at the second time point, the difference between the fresh weight of shoots in the two treatments was smaller than that at the first sampling point. However, the fresh and dry weights of the fourth leaf in the Zn deficiency treatment were significantly lower than those of the control treatment. Nutritional deficiency can accelerate flowering [19]; Arabidopsis flowering increases stem size and leaf number [20], and significant inhibition of vegetative growth is associated with an increase in the number of leaves in Brachypodium [21]. Plants adjust their allocation of leaf area and biomass accumulation to optimize nutrient utilization efficiency in the shoot and maintain photosynthesis [22]. Therefore, the reduction in total leaf biomass and dry weight in the shoots of rice treated with Zn deficiency has a special relationship with the increase in leaf number.

The rice root system is a whisker root system. After rice seeds germinate, the radicle extends down to form seed roots with only one radicle [23]. In this study, seed root senescence was inevitable, and the seed roots only had a transitional function. With rice seedling development, seed root growth and function weaken, and the seeds gradually withered [24]. The effect of Zn deficiency on the seed root was primarily reflected in the total biomass, and the total biomass of seed roots decreased faster in the Zn deficiency rice seedlings. Nodal roots, or adventitious roots that grow from the base of the plant stem nodes (including tiller nodes) are the primary parts of the rice root system. In addition, node roots provide mechanical stability to plants [25]. In Zn-deficient soils, reduced or absent rhizomes adversely affect nutrient uptake in various crops, further increasing yield losses [26]. Therefore, understanding the changes in the distribution of node roots in response to Zn in rice is crucial. During Zn deficiency, the number and length of rice nodes were significantly reduced, and a positive correlation between nodal root number and nutrient uptake (including N, P, Fe, and Zn) has been found in rice and wheat [27]. These results indicate a strong positive correlation between root elongation, development, and nutrient uptake. In this study, the number of root nodes in Zn-deficient rice seedlings did not change, consistent with the inference of Sahand Amini et al that node root development has a specific Zn requirement [21].

Zn deficiency significantly reduced Zn ion concentration and content in the leaves. From day 7 to 21, the fourth leaf was in senescence, and the biomass decreased. Under normal aging conditions, the transfer rate of Zn ions in the fourth leaf is slow. However, when affected by Zn deficiency, the aging rate, and Zn ion transfer were accelerated. However, this accelerated Zn ion transfer cannot simply be regarded as a result of Zn deficiency because leaf senescence is not a simple, continuous, and unregulated death process, and leaves undergo stringent and orderly changes during aging [28–30]. These changes are divided into several stages (primarily the initiation, reorganization, and termination stage) [31]. The regulation of leaf senescence at different stages shows different physiological and biochemical characteristics, which is supported by the results of the gene enrichment analysis in this study. Thus, although Zn deficiency leads to accelerated Zn ion transfer, it also accelerates aging. In other words, Zn deficiency may not directly lead to the accelerated transfer rate of Zn ions. Still, the accelerated transfer may be due to the accelerated aging rate during Zn deficiency, which causes the aging process of Zn deficiency-treated leaves to be faster than that of normal aging leaves.

Under Zn deficiency treatment, compared with the fourth leaf at the first time point, the biomass at the second time point increased significantly, but the concentration of Zn ions decreased, likely because the roots could not obtain Zn ions from the nutrient solution. In addition, Zn deficiency reduces the total biomass and dry weight of leaves and accelerates leaf senescence and nutrient transfer rates [32]. However, the fourth leaf was still accumulating biomass at the first two time points, but the Zn ion content did not increase. We speculate that this may be an active measure formed by rice to cope with Zn-deficiency stress. Rice optimizes the efficiency of nutrient utilization, adjusts its nutrient distribution [22], and prioritizes Zn ions and other nutrients in the latest leaves to ensure normal growth, reduce the damage caused by Zn deficiency stress, and maintain photosynthesis. However, the latest leaves did not receive sufficient nutrients, leading to shorter growth cycles and smaller leaf areas. The sample was taken when the fourth leaf was fully deployed and still accumulating biomass; therefore, owing to the growth demand of the latest leaf (the fifth leaf), the Zn ions of the last leaves would be supplied to the fifth leaf, while the supply to the fourth leaf would decrease to a certain extent. Additionally, 299 TFS from different families were identified during normal aging (C2 vs. C4), including WRKY, NAC, MYB, bHLH, and bZIP. During Zn-deficient senescence (C2 vs. D4), 348 TFS from different families were present, such as WRKY, NAC, MYB, bHLH, and bZIP.

We observed that Zn deficiency accelerates leaf aging. Leaf senescence is a necessary process that affects crop yield and quality [33]. When nutrients in the soil are too low, old leaves become a source to support the growth of new organs [34], and retransferring nutrients is particularly important at this time. Usually, nutrient transfer during leaf senescence in younger plant organs helps to improve nutrient utilization efficiency [35]. However, retransferring trace elements from leaves has received much less attention than the large amounts of elements in crops, especially when the leaves are subject to nutrient deficiencies and crops do not always contain sufficient amounts of these trace elements to meet the dietary requirements of humans. In Arabidopsis, the contents of some trace elements such as Cu, Mo, and Zn were found to decrease by < 40% during the leaf aging process, suggesting that these nutrients are mobilized from the aging leaves [36]. Arabidopsis AtbZIP19 and AtbZIP23 are the best-studied central regulators that coordinate the Zn deficiency response in plants [37]. In rice, OsbZIP48 (basic leucine zipper 48) has the highest similarity to AtbZIP19 and AtbZIP23, key transcription factors in the Arabidopsis Zn deficiency response. OsbZIP48 is a conserved functional homolog of AtbZIP19 and AtbZIP23 [34]. OsbZIP48 is highly expressed in rice seedling leaves [38], and deleting OsbZIP48 can lead to growth arrest and even death during early development [39]. However, in the present study, OsbZIP48 was not regulated under normal or Zn-deficient aging conditions. Lilay et al (2020) investigated the role of OsbZIP48 in response to Zn deficiency in rice roots and found that this gene did not respond to Zn deficiency. Therefore, some F-bZIPs induced by Zn deficiency in wheat can achieve sustained adaptive responses in long-term Zn deficiency with little transcriptional induction during Zn deficiency [40]. We found that eight genes were involved in Zn ion transfer during leaf aging in normally aging leaves, and four of them belonged to the ZIP family (OsZIP5, OsZIP9, OsZIP1, and OsZIP3). However, in contrast to normal aging, three additional genes (OsZIP4, OsZIP10, and OsZIP7) were expressed in addition to the eight genes involved in Zn ion transfer during Zn deficiency.

Sixteen ZIP transporters have been identified in rice [41], and seven ZIP genes were upregulated in this study. OsZIP1, OsZIP3, OsZIP4, OsZIP5, OsZIP8, and other family members transport Zn and Fe [42, 43]. OsZIP3, OsZIP7, and OsHMA2 are responsible for the preferential distribution of Zn in developmental tissues [12]. OsZIP1 is a Zn uptake transporter that induces gene expression under Zn deficiency [44, 45]. OsZIP1 was upregulated in Zn-deficient and normal aging leaves in this study. OsZIP1 may transfer Zn ions during leaf senescence; however, ZIP1 overexpression may reduce Zn, Cu, and Cd concentrations in rice [45]. Similar to OsZIP1, OsZIP5 is upregulated during Zn deficiency and normal leaf senescence; however, OsZIP5 may play a greater role in transporting metal ions than OsZIP1, which may be related to OsZIP5 transporting a variety of divalent metal ions. OsZIP5 expression increased in shoots and roots when induced by Zn deficiency. However, Fe and Mn deficiency only induces increased expression levels in the roots [46]. OsZIP9 was primarily expressed in the root system, with minimal expression in other parts [47]; however, OsZIP9 was upregulated in the three groups compared to the present study. OsZIP9 is a major influx Zn transporter in outer epidermal cells, and its overexpression significantly increases Zn and cadmium (Cd) accumulation in aboveground tissues and grains [48–50], indicating that OsZIP9 might be involved in Zn ion transfer during leaf senescence.

OsZIP7 is indispensable in transporting Zn and Cd from woody parts to developmental tissues and grains of rice [51]. Furthermore, during Zn deficiency, OsZIP7 is upregulated in buds [52]. OsZIP7 overexpression resulted in constant Zn and Cd concentrations in roots and nodes but decreased concentrations in stems and seeds [53, 54]. In this study, OsZIP7 was upregulated only in Zn-deficient senescence and not in normal senescence. This finding suggests that the function of OsZIP7 in transporting Zn ions during leaf senescence may be upregulated only during Zn deficiency. As a homologous gene of OsZIP7, OsZIP10 can also be strongly induced by Zn deficiency during rice leaf senescence and participates in Zn ion retransfer, which may be expressed higher than OsZIP7. In addition, although OsZIP10 was not upregulated during normal aging, OsZIP10 function loss decreased Zn and iron concentrations in rice grains [54]. Therefore, OsZIP10 could play a higher function in transferring Zn ions during the senescence of Zn-deficient leaves.

OsZIP3 was upregulated in normally aged leaves in this study, indicating that it can transfer Zn ions during leaf aging. In addition, OsZIP3 was upregulated during leaf aging with Zn deficiency; however, its upregulation during aging with Zn deficiency may not be simply induced by Zn deficiency. Although OsZIP3 is only related to Zn allocation, OsZIP3 is not induced by Zn deficiency and high Zn and is not involved in Zn absorption and transport [43]. OsZIP3 upregulation in Zn-deficient leaves during senescence may be due to enhancing senescence by Zn deficiency stress. OsZIP3 is a vital transporter that regulates Zn distribution, but it cannot transport Zn alone, and heavy metal ATPase 2 is required [43].

OsZIP4 is only related to Zn transport and redistribution in rice and is primarily expressed in the vascular bundle and mesophyll cells of leaves, phloem of stems and roots, and meristem and roots [42]. In this study, OsZIP4 expression was not upregulated in typical aging leaves but was upregulated in the other two groups, indicating that OsZIP4 transferring Zn ions in leaves may require inducing Zn deficiency. In addition, OsZIP4 expression was increased in old and new leaves under Zn deficiency, but the expression level was higher in new leaves [42]. Therefore, OsZIP4 was more likely to be expressed in young leaves under Zn-deficient conditions.

In summary, zinc deficiency inhibited the growth and development of rice shoots and roots to varying degrees. In zinc deficiency treatment, the senescence rate and zinc ion transport rate of leaves were greatly accelerated. In addition, through RNA sequencing analysis, zinc deficiency regulated more BPs, and zinc ion transport BP was particularly obvious. In zinc-deficient treatment, more ZIP family members regulated the function of transporting zinc ions during leaf aging, and OsZIP4, OsZIP10 and OsZIP7 were only up-regulated in zinc-deficient treatment. Future studies are needed to validate and explore the potential information of zinc ion transport genes in leaves.

Author Contributions

Jun Ma and Yangming Ma designed the research. Jun Ma supervised the research. Yangming Ma, Yanfang Wen, and Cheng Wang performed the experimental work and acquired the data. Yangming Ma wrote the first manuscript. Yangming Ma, Yanfang Wen, and Cheng Wang, Ziniu Wu, and Xiaojuan Yuan analyzed the data.

Yangming Ma, Ying Xiong, Kairui Chen, Limei He, Yue Zhang, Zhonglin Wang, and Leilei Li revised the manuscript. Zhaojun Liu, Zhiyuan Yang, Yongjian Sun, and Zhongkui Chen provided helpful suggestions for the experimental work. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Research Program Foundation of Key Laboratory of Sichuan Province (2022ZDZX0012); the Key Research and Development program of Chengdu City (2022-YF09-00040-SN); the Rice Breeding Project Foundation of Sichuan Provincial Science and Technology Department (2021YFYZ0005).

Data Availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Competing Interests

The authors declare no competing interests.

Thiébaut N, Hanikenne M (2022) Zinc deficiency responses: bridging the gap between Arabidopsis and dicotyledonous crops. J Exp Bot 73(6):1699–1716
Huang S, Yamaji N, Feng MJ (2022) Zinc transport in rice: how to balance optimal plant requirements and human nutrition. J Exp Bot 73(6):1800–1808
Skalny AV, Aschner M, Tinkov AA (2021) Zinc. Adv Food Nutr Res 96:251–310
Hussain A, Jiang W, Wang X, Shahid S, Saba N, Ahmad M, Dar A, Masood SU, Imran M, Mustafa A (2022) Mechanistic Impact of Zinc Deficiency in Human Development. Front Nutr 9:717064
Natasha N, Shahid M, Bibi I, Iqbal J, Khalid S, Murtaza B, Bakhat HF, Farooq ABU, Amjad M, Hammad HM, Niazi NK, Arshad M (2022) Zinc in soil-plant-human system: A data-analysis review. Sci Total Environ 808:152024
Noulas C, Tziouvalekas M, Karyotis T (2018) Zinc in soils, water and food crops. J Trace Elem Med Biol 49:252–260
Shaoxia W, Meng L, Xiaoyuan Z, Peiwen F, Yanlong C, Jianglan S, Xiaohong T (2019) Impact of foliar and root application of phosphorus on zinc concentration of winter wheat grown in China. Crop Pasture Sci 70(6):499–508
Read TL, Doolette CL, Li C, Schjoerring JK, Kopittke PM, Donner E, Lombi E (2020) Optimising the foliar uptake of zinc oxide nanoparticles: Do leaf surface properties and particle coating affect absorption? Plant Physiol 170(3):384–397
Tan L, Qu M, Zhu Y, Peng C, Wang J, Gao D, Chen C (2020) ZINC TRANSPORTER5 and ZINC TRANSPORTER9 Function Synergistically in Zinc/Cadmium Uptake. Plant Physiol 183(3):1235–1249
Palmgren MG, Clemens S, Williams LE, Krämer U, Borg S, Schjørring JK, Sanders D (2008) Zinc biofortification of cereals: problems and solutions. Trends Plant Sci 13(9):464–473
Yamaji N, Ma JF (2017) Node-controlled allocation of mineral elements in Poaceae. Curr Opin Plant Biol 39:18–24
Tan L, Zhu Y, Fan T, Peng C, Wang J, Sun L, Chen C (2019) OsZIP7 functions in xylem loading in roots and inter-vascular transfer in nodes to deliver Zn/Cd to grain in rice. Biochem Biophys Res Commun 512(1):112–118
Zhang J, Wang S, Song S, Xu F, Pan Y, Wang H (2019) Transcriptomic and proteomic analyses reveal new insight into chlorophyll synthesis and chloroplast structure of maize leaves under zinc deficiency stress. J Proteom 199:123–134
Leng Y, Ye G, Zeng D (2017) Genetic Dissection of Leaf Senescence in Rice. Int J Mol Sci 18(12):2686
Li Z, Su D, Lei B, Wang F, Geng W, Pan G, Cheng F (2015) Transcriptional profile of genes involved in ascorbate glutathione cycle in senescing leaves for an early senescence leaf (esl) rice mutant. J Plant Physiol 176:1–15
Kim T, Kang K, Kim SH, An G, Paek NC (2019) OsWRKY5 Promotes Rice Leaf Senescence via Senescence-Associated NAC and Abscisic Acid Biosynthesis Pathway. Int J Mol Sci 20(18):4437
Roy C, Kumar S, Ranjan RD, Kumhar SR, Govindan V (2022) Genomic approaches for improving grain zinc and iron content in wheat. Front Genet 13:1045955
Nouet C, Charlier JB, Carnol M, Bosman B, Farnir F, Motte P, Hanikenne M (2015) Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri. J Exp Bot 66(19):5783–5795
Kolár J, Senková J (2008) Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana. J Plant Physiol 165(15):1601–1609
Chen X, Ludewig U (2018) Biomass increase under zinc deficiency caused by delay of early flowering in Arabidopsis. J Exp Bot 69(5):1269–1279
Amini S, Arsova B, Gobert S, Carnol M, Bosman B, Motte P, Watt M, Hanikenne M (2021) Transcriptional regulation of ZIP genes is independent of local zinc status in Brachypodium shoots upon zinc deficiency and resupply. Plant Cell Environ 44(10):3376–3397
Smith DD, Sperry JS, Adler FR (2017) Convergence in leaf size versus twig leaf area scaling: do plants optimize leaf area partitioning? Ann Bot 119(3):447–456
Wang Q, Zhu Y, Zou X, Li F, Zhang J, Kang Z, Li X, Yin C, Lin Y (2020) Nitrogen Deficiency-Induced Decrease in Cytokinins Content Promotes Rice Seminal Root Growth by Promoting Root Meristem Cell Proliferation and Cell Elongation. Cells 9(4):916
Wang SJ, Ho CH, Chen HW (2011) Rice develop wavy seminal roots in response to light stimulus. Plant Cell Rep 30(9):1747–1758
Liu Z, Giehl RFH, Hartmann A, Hajirezaei MR, Carpentier S, Wirén N (2020) Seminal and Nodal Roots of Barley Differ in Anatomy, Proteome and Nitrate Uptake Capacity. Plant Cell Physiol 61(7):1297–1308
Forde BG (2014) Nitrogen signalling pathways shaping root system architecture: an update. Curr Opin Plant Biol 21:30–36
Subedi SR, Sandhu N, Singh VK, Sinha P, Kumar S, Singh SP, Ghimire SK, Pandey M, Yadaw RB, Varshney RK, Kumar A (2019) Genome-wide association study reveals significant genomic regions for improving yield, adaptability of rice under dry direct seeded cultivation condition. BMC Genomics 20(1):471
Woo HR, Kim HJ, Lim PO, Nam HG (2019) Leaf Senescence: Systems and Dynamics Aspects. Annu Rev Plant Biol 70:347–376
Kim J, Kim JH, Lyu JI, Woo HR, Lim PO (2018) New insights into the regulation of leaf senescence in Arabidopsis. J Exp Bot 69(4):787–799
Jan S, Abbas N, Ashraf M, Ahmad P (2019) Roles of potential plant hormones and transcription factors in controlling leaf senescence and drought tolerance. Protoplasma 256(2):313–329
Holland V, Koller S, Lukas S, Brüggemann W (2016) Drought- and frost-induced accumulation of soluble carbohydrates during accelerated senescence in Quercus pubescens. Trees 30:215–226
Zeng H, Zhang X, Ding M, Zhang X, Zhu Y (2019) Transcriptome profiles of soybean leaves and roots in response to zinc deficiency. Physiol Plant 167(3):330–351
Zhang Q, Xia C, Zhang L, Dong C, Liu X, Kong X (2018) Transcriptome Analysis of a Premature Leaf Senescence Mutant of Common Wheat (Triticum aestivum L). Int J Mol Sci 19(3):782
Abdallah M, Dubousset L, Meuriot F, Etienne P, Avice JC, Ourry A (2010) Effect of mineral sulphur availability on nitrogen and sulphur uptake and remobilization during the vegetative growth of Brassica napus L. J Exp Bot 61(10):2635–2646
Maillard A, Diquélou S, Billard V, Laîné P, Garnica M, Prudent M, Garcia-Mina JM, Yvin JC, Ourry A (2015) Leaf mineral nutrient remobilization during leaf senescence and modulation by nutrient deficiency. Front Plant Sci 6:317
Himelblau E, Amasino RM (2001) Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence. J Plant Physiol 158(10):1317–1323
Lilay GH, Persson DP, Castro PH, Liao F, Alexander RD, Aarts MGM, Assunção AGL (2021) Arabidopsis bZIP19 and bZIP23 act as zinc sensors to control plant zinc status. Nat Plants 7(2):137–143
Lilay GH, Castro PH, Guedes JG, Almeida DM, Campilho A, Azevedo H, Aarts MGM, Saibo NJM, Assunção AGL (2020) Rice F-bZIP transcription factors regulate the zinc deficiency response. J Exp Bot 71(12):3664–3677
Burman N, Bhatnagar A, Khurana JP (2018) OsbZIP48, a HY5 Transcription Factor Ortholog, Exerts Pleiotropic Effects in Light-Regulated Development. Plant Physiol 176(2):1262–1285
Evens NP, Buchner P, Williams LE, Hawkesford MJ (2017) The role of ZIP transporters and group F bZIP transcription factors in the Zn-deficiency response of wheat (Triticum aestivum). Plant J 92(2):291–304
Chen WR, Feng Y, Chao YE (2008) Genomic analysis and expression pattern of OsZIP1, OsZIP3, and OsZIP4 in two rice (Oryza sativa L.) genotypes with different zinc efficiency. Russ J Plant Physl 55(3):400–409
Ishimaru Y, Suzuki M, Kobayashi T, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2005) OsZIP4, a novel zinc-regulated zinc transporter in rice. J Exp Bot 56(422):3207–3214
Sasaki A, Yamaji N, Mitani-Ueno N, Kashino M, Ma JF (2015) A node-localized transporter OsZIP3 is responsible for the preferential distribution of Zn to developing tissues in rice. Plant J 84(2):374–384
Bashir K, Ishimaru Y, Nishizawa N (2012) Molecular mechanisms of zinc uptake and translocation in rice. Plant Soil 361:189–201
Ramesh SA, Shin R, Eide DJ, Schachtman DP (2003) Differential metal selectivity and gene expression of two zinc transporters from rice. Plant Physiol 133(1):126–134
Lee S, Jeong HJ, Kim SA, Lee J, Guerinot ML, An G (2010) OsZIP5 is a plasma membrane zinc transporter in rice. Plant Mol Biol 73(4):507–517
Yang M, Li Y, Liu Z, Tian J, Liang L, Qiu Y, Wang G, Du Q, Cheng D, Cai H, Shi L, Xu F, Lian X (2020) A high activity zinc transporter OsZIP9 mediates zinc uptake in rice. Plant J 103(5):1695–1709
Huang S, Sasaki A, Yamaji N, Okada H, Mitani-Ueno N, Ma JF (2020) The ZIP Transporter Family Member OsZIP9 Contributes To Root Zinc Uptake in Rice under Zinc-Limited Conditions. Plant Physiol 183(3):1224–1234
Tan L, Qu M, Zhu Y, Peng C, Wang J, Gao D, Chen C (2020) ZINC TRANSPORTER5 and ZINC TRANSPORTER9 Function Synergistically in Zinc/Cadmium Uptake1. Plant Physio 183(3):1235–1249
Zhan J, Zou W, Li S, Tang J, Lu X, Meng L, Ye G (2022) OsNAC15 Regulates Tolerance to Zinc Deficiency and Cadmium by Binding to OsZIP7 and OsZIP10 in Rice. Int J Mol Sci 23(19):11771
Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2006) Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. Plant J 45(3):335–346
Tan L, Zhu Y, Fan T, Peng C, Wang J, Sun L, Chen C (2019) OsZIP7 functions in xylem loading in roots and inter-vascular transfer in nodes to deliver Zn/Cd to grain in rice. Biochem Bioph Res Co 512(1):112–118
Gindri RG, Navarro BB, da Cruz Dias PV, Tarouco CP, Nicoloso FT, Brunetto G, Berghetti ÁLP, da Silva LOS, Fett JP, Menguer PK, Ricachenevsky FK (2020) Physiological responses of rice (Oryza sativa L.) oszip7 loss-of-function plants exposed to varying Zn concentrations. Physiol Mol Biol Pla 26(7):1349–1359
Lilay GH, Castro PH, Guedes JG, Almeida DM, Campilho A, Azevedo H, Aarts MGM, Saibo NJM, Assunção AGL (2020) Rice F-bZIP transcription factors regulate the zinc deficiency response. J Exp Bot 71(12):3664–3677

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

ZIP genes are involved in the retransfer of zinc ions during the senescence of zinc-deficient rice leaves

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Plant material and growth conditions

Ion analysis

RNA sequencing

Data analysis

RESULTS

Zn deficiency inhibits rice seedling shoot growth and development

Zn deficiency affects the growth of the fourth rice leaf

Zn deficiency alters the root architecture of rice seedlings

Zn deficiency affects Zn ion transfer during senescence in the fourth rice seedling leaf

Transcriptome response to Zn deficiency

Biological pathway involvement in Zn deficiency treatment

Zn ion transport genes are regulated differently under different conditions

DISCUSSION

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1