Activities of starch synthetic enzymes and contents of endogenous hormones in waxy maize grains subjected to post-silking water deficit

Rainfed maize in Southern China and frequently suffer water deficit at later plant growth periods. A pot trial in 2014–2015 was conducted to study the effects of drought stress (the relative soil moisture contents are 70–80% and 50–60% under control and water deficit conditions, respectively) after pollination on grain filling and starch accumulation, activities of starch synthetic enzymes, and contents of indole-3-acetic acid (IAA) and abscisic acid (ABA), with Suyunuo5 as test material. The grain fresh weight, volume, and dry weight were not affected by drought before 10 days after pollination but were restricted thereafter. The reduction at maturity was reduced by 33.3%, 40.0%, and 32.3% in 2014 and by 21.7%, 24.3%, and 18.3% in 2015. The grain filling rate was suppressed by water deficit, whereas grain moisture and starch content were slightly affected. The starch accumulation was decreased by 33.5% and 20.0% at maturity in 2014 and 2015, respectively. The activities of starch synthetic enzymes (sucrose phosphate synthase, sucrose synthase, ADP-glucose pyrophosphorylase, soluble starch synthase, and starch branching enzyme) were downregulated by post-silking drought. The ABA content was increased, whereas IAA content was decreased when plants suffered water deficit during grain filling. In conclusion, post-silking water deficit increased ABA content, decreased IAA content, and weakened the activities of starch synthetic enzymes, which suppressed grain development and ultimately reduced grain weight.

Drought stress is a main environmental constraint that restrict crop productivity worldwide. Maize are prone to drought as they are mostly grown under rainfed conditions. The annual loss of maize yield caused by water deficit dominates all environmental stresses, and the incidence is acute with increasing intensity and frequency 1 .
Grain filling duration and rate are crucial to final grain weight 2 . Studies have demonstrated that drought stress at early grain development disturb the cell division and differentiation processes required for organ establishment and starch biosynthesis 3,4 . Starch is the foremost grain component that determines grain weight, and subject to water deficit after flowering stage is, in most cases, associated with the reduced starch accumulation 5 . Water deficit occurring at initially grain development stage curtails the grain sink potential by reducing the number of endosperm cells and amyloplasts formed, thus reducing grain weight by decrease the endosperm cells to deposite starch, in terms of both duration and rate 6,7 . Studies reported that postanthesis mild drought stress shortened the grain filling period and increased grain filling rate and starch accumulation rate by enhancing the activities of starch synthetic enzymes (sucrose synthase (SuSy), ADP Glc pyrophosphorylase (AGPase), soluble starch synthase (SSS), and starch branching enzyme (SBE)), whereas the activities of those starch synthetic enzymes were weakened under severe drought stress [8][9][10] . The downregulation of AGPase activity at both the protein and transcript levels under water deficit conditions during grain filling is responsible for the significant decrease in starch deposition 11,12 . The sorghum grain filling rate was reduced by water shortage at the flowering stage due to the reduced activities of SSS, SBE, and granule-bound starch synthase 13 .
The grain endogenous hormones such as indole-3-acetic acid (IAA) and abscisic acid (ABA) significantly affect the rate and duration of grain filling 14 . Drought stress during grain filling increases the ABA content, which induces the increase of the rate and early suspension of duration 15 . Yang and Zhang 2 found that high grain ABA content under moderately soil-dried condition increased the rate, whereas too high ABA content under severely soil-dried condition reduced the rate of grain filling. Application of exogenous ABA at middle grain filling stages could regulate sink activity, grain weight was noticeably reduced with high ABA spraying concentration and mildly increased with low concentration 16 . Guoth et al. 17 found that drought-tolerant wheat genotypes only exhibited higher ABA content at early grain filling, and the sensitive cultivars maintained high ABA levels in the later grain filling stages, which led to a decreased grain yield. Lv et al. 18 and Liu et al. 19 observed that post-anthesis water stress decreased the wheat grain weight and grain filling rates due to the decrease of grain IAA and ABA contents at the early and middle stages of grain filling. Our early studies reported that waxy maize flour and starch physiochemical properties were changed by drought after pollination 20,21 . Starch formation and accumulation were co-adjusted by a series of starch synthetic enzymes and endogenous hormones, and clarfying the effects of post-silking water deficit on those enzymes and hormones could improve our understanding on starch development under drought conditions. In the present research, the grain filling and starch accumulation were clarified, the activities of starch synthetic enzymes and contents of endogenous hormones (ABA and IAA) in response to post-silking water deficit were studied. The results may offer a reference for drought-stressed starch formation of rainfed waxy maize that undergo water deficit after silking.

Results
Grain filling. Water deficit did not affect the grain fresh weight, volume, and dry weight before 10 days after pollination (DAP), but they were restricted thereafter. The reduction gradually increased with grain development. At maturity (40 DAP), the grain fresh weight, volume, and dry weight were reduced by 33.3%, 40.0%, and 32. Starch accumulation. The grain starch concentration (mg g −1 ) was slightly affected by water deficit throughout the grain filling in both years (Fig. 2). The starch accumulation (starch content × grain dry weight, mg grain −1 ) was not affected by drought before 15 DAP, and the value was reduced by water deficit thereafter, similar to the reduction of grain dry weight. At maturity, the reduction of starch accumulation under drought was 33.5% and 20.0% in 2014 and 2015, respectively. ABA and IAA content. The grain ABA content gradually increased with grain development, and it was increased by drought throughout the grain filling (Fig. 5). The IAA content in 2015 was decreased by drought, the value in 2014 was not affected at 15 DAP and reduced at the other stages, and the decrease was severe after 15 DAP in both years.

Discussion
In the present study, the grain weight (fresh or dry), volume, and average grain filling rate were decreased by post-silking drought stress. The severe reduction of in 2014 may be due to the high temperature and low sunlight duration (Post-silking day/night temperature and sunlight were 29.0/22.0 °C and 126 h in 2014 and were 28.2/21.5 °C and 145 h in 2015, respectively. Data available on http://www.tianqihoubao.com/lishi/yangzhou. html), as those heat and sunlight stresses also reduce grain weight 1 . The small volume and low weight of grains under water stress may be caused by the reduced endosperm cell numbers and few formed amyloplasts, which induced the decrease of grain sink potential 6 . The reduced rate of grain filling under drought condition induced the low grain weight, as it could serve as an indirect selection criterion for grain filling rate 22 . Grain moisture content declines during the entire grain filling, and final grain weight is achieved at values closed to 35% and moderate stress conditions affecting plant development seem to have little impact over this value 23,24 . The similar grain moisture content under control (36.3% and 34.7% in 2014 and 2015) and drought (35.3% and 32.2% in 2014 and 2015) conditions indicated semblable grain filling progress, similar observations were also reported on fresh waxy maize 21 and normal maize 25 , while extreme drought stress shortened this duration 25 . Studies on wheat and rice revealed that mild drought after anthesis shortened the duration but increased the rate of grain filling, whereas severe water shortage reduced both the rate and duration [8][9][10] . The possible explanation for this phenomenon may be due to the similar reduction of both source and sink potential in response to post-silking water deficit 21 , and the value mainly different among genotypes and little affected by moderate stresses 23,24 . Therefore, further study different water stress levels on maize grain filling may help clarify this different response.
Grain development is dependent on many factors, including sucrose availability and the activities of enzymes involved in grain starch and sugar metabolism 26 . Drought during grain filling restricted the starch deposition were reported in many crops 5 . We observed that the grain starch concentration was not affected (only reduced by www.nature.com/scientificreports www.nature.com/scientificreports/ 1.8% and 2.2% at maturity in 2014 and 2015, respectively), but the starch accumulation was severely depressed (reduction was 33.5% and 20.0% at maturity in 2014 and 2015, respectively) by post-silking water deficit, and the severe reduction of starch accumulation in 2014 may be due to the severe reduction of grain dry weight (32.3% and 18.3% in 2014 and 2015, respectively). Similar starch concentration between control and drought stress was observed on fresh waxy maize 21 and normal maize 27 . A field study on normal maize 28 and sorghum 29 also observed that starch content was slightly affected by restrict irrigation. The starch accumulation was remarkably reduced by drought, which may caused by the weakened activities of starch synthetic enzymes (SPS, SuSy, AGPase, SSS, and SBE), this opinion was also demonstrated at protein, transcript, and enzymology levels when plants suffered drought at late growth stages in wheat 10-12 and sorghum 13 .
ABA and IAA are two important endogenous hormones that significantly affect the grain endosperm cell division and expansion, grain size initiation, and grain filling rate and duration 30 . High IAA content can promote endosperm cell division and increase sink potential, which accelerate endosperm cell propagation and grain filling 31,32 . ABA is involved in the metabolic activity of key enzymes in sucrose decomposition and starch biosynthesis and regulates the process of grain weight formation 16 . Drought stress increases the amounts of ABA and  www.nature.com/scientificreports www.nature.com/scientificreports/ affects the embryo mutation, grain development, and grain components [33][34][35] . The reduced IAA content under drought was also observed in rice and caused spikelet sterility 36 . In the present study, the increased ABA content and decreased IAA content under post-silking drought stress restricted grain filling, resulted the low grain weight,