Natural Amelioration of Mn-induced Chlorosis Facilitated by Mn Down-regulation, Ammonium and Rainwater in Sugarcane Seedlings

We had previously reported that manganese (Mn)-induced chlorosis is a serious problem in ratoon sugarcane seedlings grown in acidic soils. To further monitor the progression of chlorosis and elucidate the corresponding mechanism, both plant growth and nutrient status of sugarcane plants and soils were investigated in the growth seasons of ratoon cane seedlings in 2016 and 2018. The impacts of rainfall and ammonium on chlorosis were also investigated hydroponically. The results showed that the chlorotic seedlings could green in mid-summer; Mn content in the first expanded leaf decreased significantly, whereas iron (Fe) content increased significantly during the progression of greening. The leaf Mn content in the greened seedlings decreased by up to 78.1% when compared with that in the initial chlorotic seedlings. The seedlings also showed a significant increase in seedling height and weight of the expanded leaves, accompanied by a decrease in plant Mn content during the progression of greening. Moreover, young seedlings with less Mn content showed earlier greening than older seedlings with more Mn content. The exchangeable ammonium content in the soils increased significantly during the progression of greening, and the addition of 1 mM ammonium to the chlorotic seedlings resulted in a decrease in leaf Mn content by up to 80%. Furthermore, leaf SPAD value and Fe content increased by 2.0-fold and 1.4-fold, respectively, after rainwater was applied to the chlorotic seedling. These results indicate Mn-induced chlorotic seedlings can turn naturally green, and downregulation of plant Mn content, rainfall in summer, and soil ammonium contribute to the greening of chlorotic seedlings.


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1 Introduction 2 Manganese (Mn) is an essential element for normal plant growth and development, but it 3 is toxic when present in excess. Mn toxicity is a major limiting factor for crop growth and 4 yield in acidic soils, which contains an abundance of Mn 2+ [1]. Because plant roots absorb 5 Mn as a divalent cation, any factors that change its concentration in the soil solution can 6 potentially affect the accumulation of Mn in the plant [2]. The incidence of Mn toxicity 7 strongly depends on the status of nutrients in the growth medium and concentrations of 8 other elements that affect Mn absorption, translocation, and use [2]. Most of the studies 9 on the interaction between iron (Fe) and Mn in plants have reported a negative correlation 10 between Fe and Mn accumulation in the plant shoots [2]. Fe supply has a marked effect 11 on alleviating Mn toxicity in many crops. The form of nitrogen (N) also affects Mn   Province, China, where Mn-induced chlorosis in ratoon cane has been widely found in 5 these past few decades [7]. This region has a typical subtropical monsoon climate, with an 6 annual precipitation of 1200 mm. Two fields in the same area were selected to investigate 7 greening of the chlorotic seedlings and changes in the Mn and Fe contents of the plants 8 and ammonium content in the soils as the greening progressed. In the first field, chlorotic 9 seedlings, young plants (YP), bourgeoned in April, whereas in the second field, chlorotic 10 seedlings, older plants (OP), bourgeoned in January. The soils were strongly acidic, with 11 2.5, 5.8 mg·kg -1 soluble Mn and 4.0 pH for YP and OP soils, respectively. 12 The extent of chlorosis and greening was investigated on day 0, 5, 10, 25, 35, 45, and 13 60 in 2016 and day 0, 10, 40, and 58 in 2018. Simultaneously, seedling height and leaf 14 SPAD values were measured, and the leaves and soils were sampled. The chlorotic and 15 greened leaves of the YP seedlings were recorded with a camera (Canon, EOS 5D Mark 16 IV, Japan) on day 0 and 40, respectively.

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The seedling height and SPAD values of the first expanded leaf were measured with 18 a ruler and chlorophyll meter (SPAD-502Plus; Minolta, Japan), respectively. Ten 19 seedlings were measured, and the same seedlings were measured at different time points.  Soil samples were collected from 0-20 cm of the topsoil layer. Exchangeable 10 ammonium in these samples was determined with a continuous flow analytical system 11 (SEAL-AA3， SEAL， Germany), after extraction with KCl by using the method 12 reported by Ruan et al. [10].  To investigate the impact of ammonium on Mn content in the plants, 15-day-old 20 seedlings were exposed to aerated nutrient solution (pH 5.5) with 0.5 mM MnCl 2 and 0 or To prepare Mn-induced chlorotic ratoon cane seedlings in a controlled environment, 4 the plants were exposed to 1/5 strength Hoagland solution containing 0.5 mM MnCl 2 for 5 30 days. Then, the shoots were excised from the stem bases, and the roots were exposed 6 to 0.5 mM CaCl 2 solution. After the new-generation seedlings were bourgeoned from Mn 7 pre-cultured plants, the roots of the seedlings were exposed to 1/5 strength Hoagland  The first expanded leaves of the seedlings treated with -RW or +RW were photographed 18 with the camera. The contents of Mn and Fe in the leaves were determined as described 19 above.  The greening was associated with seedling age. Visible symptoms of chlorosis were 16 not observed on May 25 (day 40), 2018, in the YP seedlings, and the leaf SPAD value 17 was 45.7; however, the symptoms were observed in the OP seedlings, which showed a 18 lower SPAD value (17.5; Fig 2B). When the tracking period extended to 60 days, the 19 visible symptoms of chlorosis disappeared in the OP seedlings as well, and the SPAD 20 value increased to 46.7. A trend of early greening was observed in the YP seedlings 21 grown in 2016 (Fig 2A).

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1 Mn content in the seedlings 2 The Mn content in the plants significantly decreased as the greening progressed ( Fig 3A   3 and B). The Mn content in the leaf was greater than 500 mg·kg -1 DW on day 0, and it   (Fig 4). No significant differences in leaf weight and seedling height were 18 observed between the plants on day 0 and 10. However, a significant increase in leaf 19 weight and seedling height was detected on day 40 (Fig 4), when the leaf SPAD value 20 increased significantly (Fig 2). The changes in leaf weight and seedling height during the 21 progression of greening were the opposite of those in leaf Mn content, which decreased as 22 the greening progressed (Fig 3A). The soils showed no significant variations in both 23 available and soluble Mn contents during the study (data not shown). These results imply The Fe content in the seedlings increased significantly as the greening progressed, and the 5 greened seedlings showed higher Fe content in their leaves (Fig 5). All the chlorotic 6 seedlings contained less than 10 mg·kg -1 FW of Fe in the leaves. On day 40, 45, 58, and 7 60, Fe contents in the leaves of the greened seedlings reached 12.9, 18.5, 15.1, and 18.7 8 mg·kg -1 FW, respectively. However, the soils showed no significant differences in 9 exchangeable and soluble Fe contents during the study (data not shown). 10 Rainy season in the study area begins in early-summer, and the precipitation is 11 extremely high (Fig S). To determine the significance of rainwater for the greening of the 12 seedlings, effects of rainwater on the chlorotic ratoon seedlings were investigated (Fig 6).

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The symptoms of chlorosis were effectively alleviated, and the chlorotic leaves obviously 14 greened after the application of rainwater for 15 days (Fig 6A). The leaf SPAD value of 15 the control plants (-RW) did not vary significantly during the 15 day period of the 16 treatment, whereas the value increased significantly (2-fold), after the application of 17 rainwater (+RW; Fig 6B). The rainwater treatment did not influence Mn content in the 18 leaves ( Fig 6C). However, Fe content in the leaves increased significantly (1.4-fold) after 19 the rainwater treatment. These results suggest that Mn-induced chlorosis was alleviated 20 by an increase in plant Fe content due to rainwater. The exchangeable ammonium content in the soils increased significantly as the greening 2 progressed (Fig 7). Although the ammonium content was similar in the same soil on day 0 3 and 10, it increased significantly thereafter. The greening YP seedlings showed a sharp 4 increase in exchangeable ammonium after 10 days, whereas the late-greening OP seedling 5 showed a moderate increase. When the seedlings greened on day 40 and 58, the 6 ammonium contents in the OP and YP seedlings increased by 3.7-and 6.2-fold, 7 respectively, when compared with the ammonium content on day 0.   Mn is one of the most abundant elements on Earth, and excess Mn is toxic to plants. 6 Excess Mn induces chlorosis in ratoon cane seedlings grown in acidic soils [7]. In the 7 present study, we found that chlorotic seedlings could green in mid-summer, and this 8 greening was associated with the downregulation of Mn content in the plants, rainfall 9 containing Fe, and soil ammonium. To the best of our knowledge, this is the first study to 10 demonstrate the progression of natural greening in Mn-induced chlorotic sugarcane 11 seedlings and its association with soil and rainfall. Our findings provide the basis for an 12 early solution for the chlorosis problem by application of Fe and/or ammonium and 13 improvement of soil nitrogen and nutrient management in agricultural practices.

Downregulation of Mn in plants is involved in greening of
15 chlorotic seedlings 16 Self-regulation is an important mechanism for plants under environmental stress. For 17 example, regulation of Nramp3 has been associated with Mn resistance in rice [12]. 18 Exudation of carboxylates from roots in response to Mn has also been demonstrated as an 19 Mn tolerance mechanism in ryegrass [13]. In the present study, we found that the 20 symptoms of chlorosis completely disappeared in mid-summer in the ratoon cane 21 seedlings in a natural environment without any manmade disturbances (Fig 1); the leaf 15/24 1 SPAD values increased significantly during the progression of greening, suggesting that 2 self-regulation mechanisms for Mn resistance act during the growth of sugarcane.

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A previous study has reported the involvement of soil moisture-induced changes in 4 available Fe in the incidence of chlorosis during season alternation. However, in the 5 present study, no obvious differences in soil soluble Fe contents were observed during the 6 progression of greening. In fact, the Mn content decreased in the plants during the 7 progression of greening (Fig 3A and B). Mn content is a key determinant of the 8 progression of chlorosis in plants. Critical toxicity content of Mn in pea, soybean, cotton, 9 and sunflower is 300, 600, 750, and 5300 mg·kg -1 , respectively [2]. In this study, the 10 chlorotic seedlings showed high Mn content in their leaves, and it ranged from 266.2 11 mg·kg -1 DW to 701.0 mg·kg -1 DW (Fig 3A and B). The leaves of OP with 342.5 mg·kg -1

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DW of Mn showed chlorosis, whereas the leaves of YP seedlings with 243.2 mg·kg -1 DW 13 of Mn did not show visible symptoms of chlorosis (Fig 3B). All the greened seedlings, 14 with higher leaf SPAD values ranging from 45.7 to 49.2 (Fig 2), showed leaf Mn content growth rate after mid-summer (Fig 5). A previous study has shown that plant tolerance to 3 Mn toxicity increases with the temperature increases [16]. Fast-growing leaves form large 4 vacuoles, thereby sequestering the potentially toxic Mn [2]. However, Mn distribution to 5 the leaf cell sap decreased after the seedlings greened (Fig 3C). The Mn content in the 6 plants decreased dramatically during the progression of greening (Fig 3). This decrease 7 could be explained by the dilution effect, rather than by Mn sequestration into cell sap 8 components such as vacuoles. The results indicate that downregulation of Mn in plants by 9 fast growth is associated with the greening of chlorotic sugarcane seedlings.

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Rainfall contributed to greening of the chlorotic seedlings 11 The precipitation in the study area increased to a great extent during the progression of 12 seedling greening (Fig S). Fe content of rainwater has been determined in previous 1 seedlings, and the greened seedlings contained higher Fe content in their leaves (Fig 4). 2 On the basis of these results, we concluded that rainfall is an important factor associated 3 with the greening of chlorotic seedlings. 4 Ammonium in the soils contributed to the greening of the 5 chlorotic seedlings 6 Ammonium is a major source of inorganic N taken up by the roots of plants and the 7 dominant N form available to plants in acidic soils [23,24]; it contributes to 79% of the 8 total soil N solution [2]. Organic N is converted to ammonium by ammonifying soil 9 bacteria, which are a type of mesophilic microorganisms. Ammoniation is inhibited in 10 cold soils, and warming increases soil ammonium availability [25]. In this study, we 11 found that the ammonium content in the soil increased significantly during the 12 progression of greening of the chlorotic seedlings (Fig. 7), and the greening was 13 accompanied by an increase in atmospheric temperature (Figs 2) [6]. Ammonium in the 14 medium had a negative impact on Mn accumulation by the plants (Fig 8). N forms affect 15 Mn toxicity [3,4]. A previous study showed that plants grown with ammonium had less 16 Mn content in their shoot tissue and developed no symptoms of Mn toxicity when 17 exposed to the same Mn content [2]. Arnon reported that ammonium sources decreased sugarcane seedlings. 23 In conclusion, our results indicate that the chlorotic seedlings could naturally turn green,       of ratoon cane seedlings were exposed to rainwater (+RW) or not exposed (-RW), 3 with no Mn addition for 15 days (A). Chlorophyll (B) and Mn and Fe (C) contents in 4 the first expanded leaf after the seedling roots were exposed or not exposed to 5 rainwater for 0 or 15 days. The ratoon cane seedlings bourgeoned from plants of the 6 previous growth season exposed to a nutrient solution with 0.5 mM MnCl 2 solution 7 for 30 days. ** on the same parameter indicates significant difference at the 0.01 8 level, according to the pairwise Student's t-test.