Maize Male Reproductive Organs Affected by Different Timings of Water Decit

Compared with female reproductive organs, the development of male reproductive organs was got less attention in maize because of its oversupply in amount even under water decit. Thus, a rainout shelter experiment was designed to explore the effect of different timings of water decit on pollen vitality and exterior and interior ultra-structure of pollen grains, starch particles in pollen grains, anther fresh weight, and vascular bundle number and its organizational structure in tassel pedicel. There were ve water treatments included in this study, viz. well water treatment (CK), water decit during 6- to 8- leaf stage (V 6 − 8 ), 9- to 12- leaf stage (V 9 − 12 ), 13-leaf stage to tasseling (V 13 − T ), and silking to blister (R 1 − 2 ), respectively. Results showed that the percentage of pollen grains with strong vitality decreased remarkably by 27.3–45.9% under water decits, while that of pollen grains with weak vitality increased by 27.2–34.7%. The percentage of pollen grains with no vitality was signicantly increased only when water decit occurred around silking, which was up to 8.6% for V 13 − T and 19.7% for R 1 − 2 compared with 1.0% for that of CK. Both shrunken pollen apertures (including annulus and operculum) and less starch particles might partially explain the weakened pollen vitality for water decits before tasseling. Furthermore, the assimilation ux to male reproductive organs might be restricted by the inuenced vascular bundle system under water decits before tasseling, with manifestation showing in anther fresh weight and starch particle status in pollen grains. Specically, V 9 − 12 and V 13 − T water decits delayed differentiation of vascular bundle but had no inuence on vascular bundle number, which might be one reason for their decreased anther fresh weight and less starch particles in pollen grains. Conversely, V 6 − 8 water decit signicantly decreased vascular bundle number but had no signicant inuence on anther fresh weight and starch particles in pollen grains. R 1 − 2 water decit almost had no inuences on above indicators except for pollen vitality. Overall, this research highlight that male reproductive organs could be inuenced by water decits in maize, which deserves more attention in further breeding especially under the background of high-quality requirement for pollen vitality of the maize hybrids that have a small tassel size.


Introduction
In maize, female reproductive organs were more prone to get fatal threat under water stress, while male reproductive organs were usually neglected mainly because of its oversupply also less in uence by water de cits [1][2][3][4] . A large tassel with more branches could cause redundant assimilation consumption and resource competition with female reproductive organs, even though it is bene t to the successful fertilization 5,6 . In order to reduce unnecessary resource competition, maize varieties with a small tassel had been regarded as one important breeding direction in the previous breeding programs [7][8][9] . In this way, a higher requirement for pollen vitality is essential since male reproductive organs were more vulnerable under unfavorable environment. Water de cit as one of the most threat abiotic stresses for crop production, it tends to be more uncertain for its occurring timing and more serious for its severity with climate change 10 . Thus, it is of great importance to explore the response of male reproductive organs to water de cit in maize, which could guide farmers and agronomist to better avoid the in uence on nal grain yield.
Water de cit at around owering could cause a great yield reduction in maize [11][12][13][14] . This decreased grain yield had be demonstrated to be mainly resulted from decreased kernel number, which was associated with the decreased amount and vitality of reproductive organs 1,2,15-17 , asynchronous owering time [18][19][20] , and zygotic abortion 4,[21][22][23] . Among these, male organs were seldomly regarded as the limitation for grain yield reduction in maize 15,24,25 . However, male organs played a crucial role for fertilization in cleistogamy crops, like rice (Oryza sativa) and wheat (Triticum aestivum L.) [26][27][28][29][30][31] . In these crops, male failure under abiotic stress might result from failed male gametophyte 32 , invalid anther dehiscence 33 , and weakened pollen vitality 34 , etc. In maize, previous researches showed water de cit around owering had limited effects on pollen vitality 2,15,24 , which might be due to (a) water de cit timing that was not overlapped with pollen sensitive period (during meiosis and mitosis) and (b) oversupply of pollen grains that might compensate for poor pollen vitality. However, there were still some documents proposed that water de cit could have direct effects on male organ growth, like, delaying male development 12 , decreasing pollen fertility 35 and pollen available amount 25,36 . Moreover, a small tassel is more favorable when breeding drought-tolerant maize hybrids that are likely more sensitive to adverse environment 9,37 . In this way, it will propose a high requirement for pollen vitality in the context of increasingly severe water de cit.
The decreased pollen vitality was related to its changed structure 38,39 , inner contents 40,41 , and endogenous phytohormones 42,43 . Speci cally, pollen abortion could be triggered by abnormal degradation of tapetum 41,44 , disturbed mitochondrial metabolism 45,46 , and abnormal dissolution of callose walls 47 . Meanwhile, the disturbed sugar metabolism in male reproductive organ was regarded as one reason for pollen sterile in several crops under water de cit 34,44,48 . Anther development and pollen maturing dependent on the photoassimilate from the source tissues. Leaf productivity could be reduced by water de cit because of stoma closure 49 and the weakened photosynthetic system 50,51 . The vascular bundle system that connects source and sink is essential for assimilate transportation and allocation, which was decreased by water de cit in both number and size 52 . The strength of sink could also have a feedback to assimilation production of source tissue 53 . Thus, the balance of "source-ow-sink" system is of great importance for male reproductive development 54,55 .
Compared to cleistogamous crops, there were few researches focusing on male reproductive organ development in maize even under water de cits. In this study, we focused on male reproductive organ development under different timings of water de cit, and explored the effect of water de cits on pollen vitality, and the possible underlying accessories. Exterior and interior ultra-structure of pollen grains, anther fresh weight, vascular bundle number and its organizational structure in tassel pedicel were observed around tasseling to explain their relationship with pollen vitality under water de cit.

Materials And Methods
Plant growing and water regimes Hybrid maize Zhengdan 958 was used as material in this study. Plants were growing in cement sealed ponds under rainout shelter during 2014-2016 at Shangzhuang Experimental Station, China Agricultural University (Beijing, China), as shown in Li et al. 52 . Plants were thinned at the 3-leaf stage and then plant density was established with 0.6 m row spacing and 0.2 m plant spacing. In order to meet the nutrient requirement for plant growth, N (60 kg ha -1 ), P (90 kg P 2 O 5 ha -1 ), K (150 kg K 2 O ha -1 ) were evenly scattered on the soil surface, and then plowed into soil by shovels before planting. Top-dressing was proceeded at the 12-leaf stage through furrowing application with 120 kg ha -1 N. During maize growth season, cutworm (Agrotis segetum) and corn borers (Ostrinia nubilalis) were controlled by applying phoxim at the 3-leaf stage and carbofuran at the 10-leaf stage, respectively. Weeds were removed arti cially when it is necessary. The plant material employed in the present is in compliance with relevant institutional, national, and international guidelines and legislation.
Five irrigation regimes were arranged with a completely randomized design, including four water de cit treatments and one well-irrigated treatment. Five-times irrigation processed during maize growth season were at 6-leaf stage (V 6 , the 1 st irrigation), 9-leaf stage (V 9 , the 2 nd irrigation), 13-leaf stage (V 13 , the 3 rd irrigation), tasseling (R 1 , the 4 th irrigation) and blister (R 2 , the 5 th irrigation), respectively, for well-irrigated treatment. Water de cits were achieved by omitting irrigation once at 6-leaf stage for V 6-8 , at 9-leaf stage for V 9-12 , at 13-leaf stage for V 13-T , and at tasseling for R 1-2 , respectively. Irrigation was applied manually by hose. The irrigation amount was calculated separately for every 20 cm soil layer (0-20 cm, 20-40 cm, and 40-60 cm) and then summed up, as shown in Li et al. 52 . The equation for irrigation amount (I) of each layer was I = 1000 BD·D·A (80% FD SWC), where BD means soil bulk density, D means soil layer depth, A means plot area, FD means eld capacity, and SWC means soil water content. The soil water content increased up to 80% of eld capacity after irrigation, while it dropped down to approx. 50% of eld capacity during water de cit period 52 . Shelter was covered whenever it rained before the end of all water de cit treatments (R2), and then it kept opening afterward. The meteorological data were collected from China Meteorological Administration (http://data.cma.cn/site/index.html), showing averaged temperature and sunshine hours during water de cit periods (Supplementary Table 1).

Sampling for pollen grains and TTC staining for pollen vitality
Fresh pollen grains were collected from each treatment whenever tassel is blooming in 2016. Target tassels were shaken gently to remove old pollen grains before sampling. And then fresh pollen grains were collected by gently taping tassels with a container covered by a ne sieve screen below. These sampled pollen grains were put into plastic bags with exhausted air, and then transferred to the lab immediately with an iced container.
Pollen vitality was detected by TTC (2,3,5-triphenyltetrazolium chloride) staining with 0.5% (w/v) TTC solution dissolved by 0.1 M PH=7.0 sodium phosphate buffer (NaHPO 4 -NaH 2 PO 4 ). Fresh pollen grains were dropped on a slide with a small spoon and mixed with one drop of TTC solution by a pipette. Specimens were covered by the coverslips and put in the dark over 15 minutes, and then observed and took microphotos under stereoscope (Olympus SZ60, Olympus Imaging China Co., Ltd., Beijing, China) immediately. At least 5 elds of view were selected for statistics and computing percentage of each type of pollen grains. Pollen grains were classi ed into 3 types according to its displayed color after TTC staining. Speci cally, pollen grains in dark red were regarded with strong vitality, in light red with weak vitality, and in ivory with no vitality, respectively. Percentage of different types of pollen grains was calculated through the number of each type of pollen grains divided by the number of all observed pollen grains.
Anther fresh weight measurement and organizational structure observation of vascular bundles in tassel pedicel Five tassels from each treatment were collected at around tasseling (55 days after planting) in 2016, and then stored at -80℃ freezer before measurement. All three strong anthers were extracted by dissecting needle from fteen stalked orets located at middle tassel in orescence, followed by fresh weight measurement immediately (Fig. 3).
Three tassels collected at 3 days after the 3 rd irrigation (49 days after planting) in 2016 were used for organizational structure observation. The observed segments were about 1 cm-long sections cut from the basal tassel pedicel through single blades. Subsequently, samples were put into formaldehyde acetic acid solution (FAA) composed of 10% formasldehyde, 50% ethanol, and 5% acetic acid immediately and stored at 4℃ before para n section proceeding. After a series of dehydrated and in ltrated, tassel pedicel segments were nally embedded in para n wax as showed in Li et al. 52 . Slices with thickness of 4 μm were acquired through Leica RM 2016 microtome (Leica Shanghai Instrument Co., Ltd. Shanghai, China). Safranin-fast green (0.5%, w/v) staining were used for slices coloring. Finally, micrographs were taken by Nikon Elipse Ci (Nikon Instruments Inc., Shanghai, China), and analyzed by Case Viewer (3DHISTECH Ltd., Utah, Hungary).
Ultra-structure observation of exterior and interior pollen grains Part of the above collected fresh pollen grains in each treatment were saved in two 2 ml centrifuge tubes lled with 2.5% glutaraldehyde dissolved by PH=7.4 0.1M PBS (sodium phosphate buffer, NaHPO 4 -NaH 2 PO 4 ). Pollen grains from one centrifuge tube was used for exterior ultra-structure observation through scanning electron microscope (Hitachi S-3400 N, Toyko, Japan) after pretreatment and coated with gold palladium, as mentioned in Wang et al. 56 . Pollen grains for interior ultra-structure observation was through transmission electron microscope (JEM-1230, Tokyo, Japan), which also referred to the method mentioned in Wang et al. 56 .

Results
Pollen vitality under water de cit As shown in Fig. 1, results displayed that well-irrigated treatment (CK) had the highest percentage of pollen grains with high vitality (61.5%), while the percentages of pollen grains with weak (37.5%) and no (1.0%) vitality were the lowest, compared to water de cit treatments (Fig. 1). V 6-8 and V 9-12 treatments hold similar percentages in all three types of pollen grains ( Fig. 1A-B, C-D). In detail, the percentages of pollen grains with a high vitality decreased down to 34.2% for V 6-8 and 31.8 % for V 9-12 , while the percentages of pollen grains with weak vitality increased up to 64.9% for V 6-8 and 65.3% for V [9][10][11][12] , as compared to that of CK. There was no signi cant difference observed for the percentages of pollen grains with no vitality among V 6-8 , V 9-12 and CK. For V 13-T and R 1-2 treatments, the percentages of pollen grains with a high vitality decreased signi cantly down to 19.2% for V 13-T and 15.6% for R 1-2 , while the percentages of pollen grains with both weak and no vitality increased signi cantly (Fig. 1A-B, E-F).
Speci cally, the percentages of pollen grains with a weak vitality were 72.2% for V 13-T and 64.7% for R 1-2 , and percentages of pollen grains with no vitality was 8.6% for V 13-T and 19.6% for R 1-2 .
Size and ultra-structure of pollen grains under water de cit The size of pollen grains had been in uenced by water de cits especially during V 9 -R 1 (Fig. 2 A 1 -D 1 , F 1 and F 2 ). Signi cant decrease in pollen area was shown for V 9-12 and V 13-T in 2015 and for V 9-12 during 2016, but increased for V [6][7][8] in 2016, compared to CK treatment. There was no signi cant difference among CK, V 6-8 , and R 1-2 in 2015, and among CK, V 13-T , and R 1-2 in 2016 ( Fig. 2 A 1 -E 1 , F 1 and F 2 ).
Although there were no consistent results between two experimental years, the annulus area of pollen aperture were prone to be shrinking after water de cits ( Fig. 2A 2 -E 2 , G 1 and G 2 ). Speci cally, the annulus area of pollen aperture had a trend to be smaller with the delay of pre-owering water de cits, with a remarkable decrease in V 6-8 , V 9-12 , and V 13-T compared to CK during 2015 ( Fig. 2 A 2 -D 2 , G 1 and G 2 ). The annulus area of pollen aperture were not signi cantly different between CK and R 1-2 in both 2015 and 2016 ( Fig. 2 A 2 , E 2 , G 1 and G 2 ).
Regarding to the operculum area of pollen aperture, signi cant decrease in V 13-T in 2015 and V 9-12 in 2016 was observed, compared to CK (Fig. 2 H 1 and H 2 ).
Anther fresh weight and starch particles in pollen grains under water de cit As shown in Fig. 3, the longer anthers from 15 orets were pooled together for anther fresh weight measurement. Results showed that anther fresh weight signi cantly decreased in V 9-12 and V 13-T treatment but was not affected in V 6-8 and R 1-2 treatment, compared to CK (Fig. 3 A). In addition, anther fresh weight decreased greater for later water de cits prior to owering, with the most decrease in V 13-T .
The effect of water de cit on starch particles in pollen grains were observed through transmission electron microscope (TEM), as shown in Fig. 4. With the same magni cation, starch particles were more and bigger in pollen grains from well-irrigated treatment than that from water de cit treatments. Starch particles in pollen grains from V 9-12 and V 13-T had obviously less number than other treatments.
Furthermore, starch particles seemed to concentrate along one side of pollen grain wall for CK and V [9][10][11][12] but scattered more evenly in pollen grains from V 6-8 , V 13-T , and R 1-2 treatments, which was because of uncontrolled crossing position when slicing.

Cross section and the vascular bundle number in tassel pedicel under water de cit
Vascular bundle number in tassel pedicel and its organizational structure were showed in Fig. 5. Water de cits signi cantly decreased the number of vascular bundles only for V 6-8 water de cit compared to well-watered treatment (Fig. 5 A-C). The size of vascular bundle showed smaller in water de cit treatments than that of CK treatment, even though there was similar number of vascular bundles among V 9-12 , V 13-T and CK (Fig. 5 A-E).

Discussion
In this study, maize pollen grains with weak vitality increased due to water de cits, while that with no vitality increased the most in R 1-2 water de cit, followed by V 13-T , and with no difference between V 9-12 and V 6-8 water de cit and CK. As a consequence, water de cit imposed around owering hold the lowest amount of pollen grains that with high vitality (Fig. 1). Pollen grains and anthers were in developmental progression before tasseling, both of which were sensitive to unfavorable environments and were more easily in uenced 35,57,58 . Water de cit had a greater in uence on the size of pollen grains, grain aperture and anther weight nearly prior to tasseling (Fig. 2, 3). During the period from V 13 to tasseling, male reproductive organ develops, including pollen grain size, pollen aperture and, and starch condition, a sensitive growth period to water de cit 35,59,60 , which can explain the smallest anther fresh weight in V 13-T water de cit (Fig. 3). The response of vascular bundle system could be one reason for the reduced anther fresh weight in pre-tasseling water de cit (Fig. 3, 5). Moreover, the reduced starch particles in pollen grains might also contribute to the low fresh weight of anthers (Fig. 3, 4). Differently, R 1-2 water de cit had no in uence on pollen characteristics, since pollen grains and anthers almost matured at around tasseling ( Fig. 1-4). The difference in pollen vitality between R 1-2 water de cit and CK might be related to the plant water status that was caused by the irrigation regime in this study. Soil moisture of R 1-2 kept dropping since irrigation at 13-leaf stage (V13) till the next water supply at around 10 days after silking. However, there were documents indicating that the water potential of reproductive organs was prone to be sustained or less in uenced despite an appreciable decrease in leaf water potential 2,35 . Thus, the indirect consequence of discrepancy in water status of plants might contribute to the different performance in pollen vitality between these two treatments such as ABA that can be regarded as a transportable sporicidal signal 35,56 .
The development of male reproductive organs was tightly associated with the assimilate ux through vascular bundle system that connecting productive and sink tissues 61 . Vascular bundle in tassel pedicel was as the terminal of assimilation ux that supporting male in orescence, which was of great essential for the assimilation amount. In this study, there were differences in the amount and status of vascular bundles in tassel pedicel among treatments during processing, even they were observed far away from its nal matured status as mentioned above (Fig. 5). Thus, the differentiation of vascular bundle largely depended on the growth stage of the whole plants. Pollen vitality as the nal manifestation of male organ development was in uenced by a series of developmental processes. Pollen vitality was closely associated with pollen structure per se. Mature pollen grains with a strong vitality were usually fully hydrated also with a shape of in ated prolate spheroid, while pollen grains were prone to be shrank after losing vitality 62 . In our study, there was no obvious sur cial structure damage by all water de cits, but shrinking size in both pollen grains itself and pollen apertures existed especially for V 9-12 and V 13-T water de cits (Fig. 2). The percentage of pollen grains with no vitality was less affected by these two water de cits, but the percentage of pollen grains with weak vitality had been signi cantly increased (Fig. 1).
Thus, we deduced that this shrinking pollen grains and pollen apertures by these water de cits might not have a fatal damage for pollen grains but it might associate with the weak vitality of pollen grains. Then, the development of anthers-the carrier of pollen grains is also of great essential for pollen maturation. In our study, water de cits at V 9-12 and V 13-T had an irreversible effect on the fresh weight of anthers that carried with smaller pollen grains ( Fig. 2 and 3). In rice, the abnormal programmed cell death happened for anther walls after suffering water de cit, which had a detrimental effect on pollen vitality 32 .
Additionally, starch content and its related sugar metabolism inside pollen grains also in uenced pollen vitality 28,34,63 . In this study, pollen grains from V 9-12 and V 13-T water de cits had less starch particles, which could explain the increased percentage of pollen grains with weak and no vitality (Fig 1 and 4). Even though water de cit could decrease leaf photosynthesis ability during and after water stress, the non-reducing sugar in pollen still increased 52,64 . Moreover, the starch in pollen grains decreased, which suggested that not carbon starvation but the process from substrate to starch was obstructed in pollen grains 28,65 . Also, Sheoran and Saini 28 showed that the invertase and starch synthase in anthers played an important role in restraining the utilization of assimilation and starch synthesis. Furthermore, the assimilation ow might be restrained by delayed development of vascular bundle in tassel pedicle in this study, as showed in Fig. 5. There was also report indicating that decreased starch in sterile pollen was accompanied with poorly developed vascular bundle system 61 . Additionally, weak pollen viability was also related to the lower moisture content because of the increased vapor pressure de cit (VPD) between air and pollen itself after exposure in air for several hours 62,66 . Overall, in uenced pollen vitality was a comprehensive results of a series factors after maize suffering water de cits.
Pollen vitality was in uenced by water de cits, in which pollen grains with high vitality decreased the most under water de cits around owering in this study (Fig. 1). This result seemed to be inconsistent with previous results 15,24,67 . The inconsistency could be explained by the different approaches used for pollen vitality testing. The reciprocal cross pollination approach that was used in previous studies could make the weak pollen vitality obscured due to the oversupply of pollen grains, while TTC staining could directly detect the vitality of each individual pollen grains 68 . The in uenced pollen vitality had no fatal effect on kernel setting under water de cits, even with 14.0-19.1% kernel number losing observed for water de cits around owering 52 . One hand, the poor quality of the pollen grains could be compensated by the great amount of pollen grains. There are over millions of pollen grains produced by each tassel, while there are maximum approx. 1000 silks for individual ear need to be fertilized 36 . The other hand, documents had clari ed other more important factors associated with this kernel number reduction under water de cits in maize, like, asynchronous owering, silk arrest, and kernel abortion 4,16,17,22 .
As mentioned above, the detecting method for pollen vitality will affect the judgement for its effect on kernel setting. Several methods including direct and indirect approaches were usually used in one experiment to improve measurement precision [69][70][71] . Merits and demerits usually coexisted in each approach. In-vitro media culture has been regarded as a more precise and reliable approach, in which pollen vitality is determined through calculating pollen germination percentage, but a high possibility of pollen bursting was the drawback of this approach 72,73 . Thus, it is urgent and necessary to develop a more e cient and burst-avoided cultivation medium. Also, pollen germination could also be observed in vitro through uorescence after aniline blue staining 29,74 . Dye staining reaction as another direct testing approach is faster and easier to operate but sometimes prone to overestimate pollen vitality 75 . The available dyes for detection include 2,3,5-triphenyltetrazolium chloride (TTC) for redox reaction, iodine/potassium iodide for starch staining (I 2 -KI), and isatin for proline staining 76,77 . Pollen vitality could also be determined through seed set counting after pollination with the tested pollen grains 77 , which is a relatively accurate approach and widely used by agronomist. But the equivalent amount of pollen grains is di cult to be determined in this approach.

Conclusion
This study clari ed that the development of male reproductive could be affected by water de cits in maize, which is worthy of more attention in further studies. Speci cally, weakened pollen vitality had been observed under water de cits especially around owering. There was also no fatal damage for pollen grains, but the shrunken pollen aperture and pollen grains itself might be associated with its weakened pollen vitality for water de cits imposed before tasseling. Less starch particles displayed in pollen grains from V 9 − 12 and V 13 − T water de cits, which could partly explain the decreased anther fresh weight also the weakened pollen vitality. Furthermore, the decreased amount of vascular bundle in tassel pedicel under water de cits may potentially limit the ux of assimilation to pollen grains. Additionally, R 1 − 2 water de cit had no in uence on above mentioned pollen characteristics except for pollen vitality indicating there are other reasons for the decreased pollen vitality in water de cit near owering.  Figure 1 The percentage of different types of pollen grains (A) and TTC staining slices of pollen grains (B-F) under well-irrigated and water de cit treatments during 2016. CK, well-irrigated treatment, V6-8, water de cit during 6-to 8-leaf stage, V9-12, water de cit during 9-to 12-leaf stage, V13-T, water de cit during 13-leaf stage to tasseling, R1-2, water de cit during silking to blister. Pollen grains in dark red means strong vitality, in light red means weak vitality, in ivory means no vitality. Different letters among treatments within same type of pollen grains represent signi cant difference at p<0.05 in the histogram.

Figure 5
Vascular bundle number (A) and its micrographs (B-E) at 49 days after planting (DAP) under well-irrigated and water de cit treatments during 2016. CK, well-irrigated treatment, V6-8, water de cit during 6-to 8leaf stage, V9-12, water de cit during 9-to 12-leaf stage, V13-T, water de cit during 13-leaf stage to tasseling, R1-2, water de cit during silking to blister. Different letters among treatments represent signi cant difference at p<0.05 in the histogram.

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