Plant senescence is the last link in the process of growth and development and is a process of programmed cell death. The decline of plant organ or whole plant function is beneficial to the survival and continuation of plant individuals, which has obvious positive significance in developmental biology, but abnormal senescence or early senescence has serious damage to plants (Ding and Wu, 2011). Plant senescence can be divided into four types: whole plant senescence, top senescence, progressive senescence and deciduous senescence. Generally, senescence first occurs in leaves, which is characterized by a significant decrease in protein content, nucleic acid content, photosynthetic rate, respiratory rate and so on, which will lead to the decrease of plant assimilation ability and crop yield (Zheng et al., 2009). Rice is an important food crop in China, as well as monocotyledoneae. The senescence of rice is generally the whole plant senescence, which often occurs after flowering and fruiting (Gan and Amasino, 1997). Many agricultural scientists have always attached great importance to the causes and molecular mechanisms of rice early senescence. It is of great significance to explore the early senescence-related genes and clarify their functions for the exploration of rice senescence mechanism and variety improvement.

At present, many leaf early senescence mutants have been identified in rice, and more than 20 rice leaf early senescence genes have completed gene mapping and partial functional analysis. These genes are distributed on 11 chromosomes except chromosome 12 (Li et al., 2020). According to the characteristics of senescence phenotype, leaf senescence mutants are divided into two types: leaf etiolation and leaf spots. Their wild type varieties (lines), premature aging traits, senescence occurrence period and target gene location are different (Li et al., 2020). Rice leaf apex dead mutant lad turned yellow and gradually died starting from tillering stage until mature. Its regulatory gene LAD was located on chromosome 11 (Du et al., 2012). The rice spotted leaf mutant spl28 began to form leaf-like lesion at flowering stage, leading to leaf early senescence. Its regulatory gene SPL28 was located on chromosome 1, encoding a clathrin associated adaptor protein complex 1, medium subunit μL (AP1M1), participating in the material transport pathway of Golgi apparatus, and its functional deficiency caused plant allergic reactions (Qiao et al., 2010). Although some rice leaf senescence regulatory genes have been cloned, their physiological and molecular regulatory mechanisms are still unclear. In the future, more regulatory genes related to leaf early senescence or senescence can be mined through map-based cloning approach, and the research on leaf senescence-related mechanism can be carried out by using technologies such as recurrent mutation and gene editing, so as to continuously enrich the regulatory network of senescence.

Rice leaf senescence will cause changes in various physiological indexes in plants, such as the decrease of photosynthesis, the degradation of soluble protein, the increase of lipid peroxide, the decrease of ATP enzyme activity, the sharp decrease of chlorophyll synthesis and so on. The plant leaf senescence will lead to the decrease of chlorophyll-protein complex and photosynthetic yield because that the photosynthetic carbon cycle in leaves is destroyed, and a variety of free radicals and membrane lipid peroxidation are accumulated in cells, resulting in the stems and leaves wilt, leaves early senescence, and poor grain filling, resulting in the decrease of yield (Tang et al., 2005).

At present, most of the rice early senescence mutants began to appear early senescence from the vegetative growth period such as seedling stage or tillering stage, and only a few mutants showed early senescence during the reproductive growth period of rice, indicates that the mechanism of early senescence is not single. The leaves of HuhuiH103 early senescence mutant psl2 turned yellow and senescence after heading and the flag leaves completely aged and died when rice was fully mature. Its regulatory gene was located on chromosome 3 (Zhang et al., 2014).

In this study, early senescence mutant es-h was screened and identified from rice EMS mutant bank, and genetic analysis and gene mapping were carried out. The mutant showed early senescence phenotype of leaf rust spots during the reproductive growth period, and its candidate gene for early senescence regulation was located on chromosome 1, which was not allelic to the reported early senescence gene locus and was a new early senescence gene. This study plays an important role in promoting the cloning of rice early senescence regulatory genes and their functional analysis, molecular regulation mechanism analysis, and the improvement of rice yield and quality.

1 Results and Analysis

1.1 Phenotype identification and agronomic traits of rice early senescence mutant es-h

Rice early senescence mutant es-h was screened from japonica rice variety Hwacheongbyeo after EMS mutagenesis. There was no significant difference in phenotype between the mutant and the wild type before heading. The es-h mutant began to show a senescence phenotype by the first week of heading: rust spots appeared at the tip of the lower leaf margin and gradually spread to the whole leaf, and then the leaf curled and died. Then similar symptoms appeared in the upper leaves. es-h mutant withered rapidly with the filling process, except for flag leaves, leaves almost all died, and the whole plant died off by the fifth week of heading (Figure 1). At heading and maturity stages, compared with the wild type, the es-h mutant plants were shorter, the number of spikelets decreased significantly, the seed setting rate and 1000-grain weight decreased significantly, but there was no significant difference in heading stage, panicle length, panicle excertion and panicle number per plant (Table 1). Results showed that the es-h mutant appeared early senescence during the reproductive period, and the whole plant senescence and died off.

1.2 Physiological characteristics of es-h mutant

In order to further clarify the early senescence physiological characteristics of es-h mutant, we determined the physiological indexes between es-h mutant and wild type leaves such as SPAD value, chlorophyll content, Fv/Fm value and soluble protein content. Results showed that the SPAD value and chlorophyll content of es-h mutant were significantly lower than those of wild type, and decreased gradually with time from heading, but the decrease of es-h was greater than that of wild type (Figure 2A; Figure 2B). The Fv/Fm value of es-h mutant was significantly lower than that of wild type (Figure 2C). The maximum photosynthetic rates of the es-h mutant and the wild type were close to 0.8 on the day of heading, and then gradually decreased. The wild type decreased slowly and remained about 0.65 after 42 d of flowering. However, the es-h mutant began to decline rapidly after 7 d of heading, decreased to about 0.6 after 14 d, and decreased to about 0.4 after 42 d, and its extent and speed were significantly higher than those of the wild type. 7 d after heading, the decreasing rate of soluble protein content of es-h mutant was significantly accelerated, which was only 2.5 mg/g at 42 d, while that of wild type was 15 mg/g (Figure 2D). These results further suggest that es-h mutant plants have physiological characteristics of early senescence compared with wild type plants.

1.3 Genetic analysis of es-h mutant

The es-h mutant was used as the female parent, and F₁ was obtained by hybridization with rice varieties Hwacheongbyeo and Milyang23, respectively. The F₂ segregation population was obtained by self-cross. The genetic analysis and gene mapping were carried out using the F₂ population. The results showed that there were 83 normal plants and 37 early senescence plants in the F₂ population of es-h mutant and its wild type (Hwacheongbyeo), with the χ2 (3:1) of 2.198 and the probability value of 0.138. In the F₂ population of es-h mutant and Milyang23, there were 255 normal plants and 72 early senescence plants, with the χ2 (3:1) of 1.871 and the probability value of 0.252 (Table 2). The results showed that the number of plants with normal phenotype and premature aging traits in the two different combinations were consistent with 3:1, indicating that the premature aging traits of es-h mutant were controlled by a pair of recessive genes.

1.4 Fine mapping of es-h mutant

In order to locate the regulatory genes of early senescence mutant es-h, STS molecular markers were used to screen the linkage markers of early senescence mutant genes in F₂ plants of es-h/Milyang23 hybrid, and these markers were used to analyze the genetic population and construct the linkage genetic map. The primary mapping results showed that the gene was located between STS markers S1134 and S1136F on the long arm of chromosome 1, and the genetic distances were 0.6 cM and 0.5 cM, respectively (Figure 3A). In order to carry out fine mapping, 2 079 F₂ and its derived F₃ plants were used for genotype and phenotype identification, respectively, by screening the offspring (F₃) of recombination and hybrid plants from the target section of F₂. Through genotype and phenotype analysis, 6 recombinant plants were finally screened, and the early senescence gene was located between S1136K and S1136S, with a physical distance of 44.2 kb (Figure 3B). There were 7 open reading frames (ORFs) with unknown functions in this region, including 4 disease resistance-related expression proteins, 1 vacuole protein sorting protein, 1 hypothetical protein and 1 unknown function expression protein (Table 3). The results of gene mapping showed that Es-h locus was non-allele with all leaf senescence genes reported at present. It can be speculated that Es-h is a new gene regulating leaf senescence.

Based on the analysis of the protein functional domains of the 7 genes, it was found that the LOC_Os01g57260 gene had VPS28 protein domain, the protein encoded by the LOC_Os01g57270 gene had NB-ARC domain and LRR protein domain, and the protein encoded by the LOC_Os01g57280, LOC_Os01g57310, LOC_Os01g57340 genes had NB-ARC domain and LRR protein domain. In which, the LOC_Os01g57310 gene was a blast resistance gene Pi37, and the protein encoded by LOC_Os01g57294 gene had an ECB2 protein domain (Table 3).

2 Discussion

Early senescence is an abnormal aging process, which has a negative impact on the normal growth and development of plants. During the rice production, early senescence of leaves and roots often occurs, causing stem and leaves to wilt, resulting in low seed setting rate, increasing empty rate, and reducing yield. Especially in the late growth stage of rice, early senescence affects grain filling and transportation and distribution of dry matter, resulting in rice yield or rice quality reduction. The es-h mutant identified in this study withered and the whole plant died off during the reproductive growth period, and its photosynthetic physiological indexes at the reproductive growth period were significantly lower than those of the wild type, showing obvious premature aging traits (Figure 1; Figure 2).

60% to 80% of the nutrients needed for rice grain filling come from leaf photosynthesis, and the main photosynthetic organ of plants is leaf (Huang and Peng, 2007). Therefore, it is of great significance for rice yield to explore the molecular mechanism of leaf growth, development and senescence (Duan et al., 1997; Wang et al., 2003; Zhang et al., 2014). Early senescence of leaves led to insufficient assimilation of photosynthates, which eventually led to an increase in the number of empty grains and a decrease in seed setting rate and 1000-grain weight. Poor vegetative growth led to the inhibition of reproductive growth and the decrease of the number of spikelets. The yield components of rice were panicle number per plant, spikelet number per panicle, seed setting rate and average 1 000-grain weight. Compared with the wild type, the yield components of es-h mutant decreased significantly except panicle number per plant, which eventually led to a decrease in yield (Table 1).

Leaf senescence is a highly programmed process, and its genetic and physiological mechanisms are very complex. The most intuitive feature of plant senescence is that the leaf color turns yellow. With the senescence of leaves, the ultrastructure of chloroplast changes obviously, the ability of photosynthesis decreases, and the protein abundance decreases (Duan et al., 1997; Lim et al., 2007; Yan et al., 2007). Compared with the wild type, the chlorophyll content of es-h mutant decreased faster, indicating that the photosynthetic capacity of the mutant decreased faster. The decrease of photosynthetic capacity will directly lead to the decrease of plant assimilation ability and the accumulation of photosynthetic products. The lower the Fv/Fm value, the greater the impact on photosynthesis of plants, the worse the health of plants under strong stress. Soluble proteins in plants are rich in enzymes involved in various metabolisms. The decrease rate of soluble protein content in es-h mutant was significantly accelerated after 7 d of heading, indicating that the metabolic ability of plants was significantly decreased (Figure 2). In a word, compared with the wild type, the es-h mutant entered the aging stage of significant decrease in photosynthetic capacity, health status and metabolic capacity earlier, showing an early senescence phenotype.

It is reported that there is no difference between the Ospse1 mutant and the wild type in the vegetative reproductive stage, while the mutant senescence rapidly after heading. OsPSE1 encodes a pectate lyase, which may regulate the early senescence of rice leaves through pectase activity (Wu et al., 2013). In this study, the early senescence phenotype and occurrence period of es-h mutant were similar to those of Ospse1 mutant, and the early senescence phenomenon and 1000-grain weight and seed setting rate of key agronomic traits related to yield were significantly reduced from the reproductive growth period. So far, most of the reported early senescence mutants have shown premature senescence from vegetative growth stages such as seedling stage or tillering stage, while only a few mutants have shown premature senescence from reproductive growth stage. Using genetic engineering methods to overexpress or knock out these premature senescence genes in reproductive growth period, to create or improve rice varieties can delay the senescence of rice in reproductive growth period, which is conducive to the improvement of rice yield and quality.

The es-h mutant was a stable strain bred by multiple generations of self-crossing after EMS mutagenesis of rice variety Hwacheongbyeo. The premature aging traits of the mutant was controlled by recessive single gene (Table 2), located between S1136K and S1136S on chromosome 1 (Figure 3). Studies have shown that 4 early senescence genes have been located on chromosome 1 of rice, namely SPL28 (Encoding the adapter protein complex), OsPSE1 (Encoding the pectate lyase), SPL33 (Encoding the eEF1A protein), and ESL-1 (SCAR protein), respectively (Qiao et al., 2010; Wu et al., 2013; Rao et al., 2015; Wang et al., 2017). es-h gene and the above 4 genes are non-allelic, so it can be inferred that es-h is a new gene regulating leaf senescence.

The physical distance of es-h gene was 44.2 kb, and there were 7 ORFs, including 4 resistance-related expression proteins (Table 3). LOC_Os01g57270 encodes disease resistance RPP13-like proteins, LOC_Os01g57280, LOC_Os01g57310, and LOC_Os01g57340 genes encodes RP1 proteins, all of which contain NB-ARC and/or LRR domains. Studies have shown that NB-ARC and/or LRR domains are mostly present in disease resistance proteins and are also involved in programmed cell death, which can cause cell senescence (Ooijen et al., 2008). It is reported that MicroRNA1916 (sly-miR1916) of Lycopersicon esculentum can regulate the expression of RPP13-like proteins, which play an important role in the response of Lycopersicon esculentum to pathogen infection (Chen et al., 2018). Whether the es-h gene located in this study encodes the resistance-related genes in the target region remains to be further studied through gene cloning.

3 Materials and Methods

3.1 Experimental materials

Rice early senescence mutant is a leaf early senescence mutant obtained by ethyl methylsulfonate (EMS) mutagenesis of the japonica rice variety Hwacheongbyeo. It has been stably inherited after successive generations of self-crossing and named as es-h (early senescence-Hwacheongbyeo). The es-h mutant was used as the female parent and hybridized with wild type Hwacheongbyeo and South Korean unified rice variety Milyang23 to obtain F₂ segregation population. And the F₂ population was used for genetic analysis and gene mapping.

3.2 Physiological index determination

30 Hwacheongbyeo and mutant plants were selected every 7 d after heading. SPAD values were measured by chlorophyll meter (SPAD meter). The same leaf was measured in the upper, middle and lower parts, respectively. The measured values were recorded, and the average value was taken as the SPAD value of this leaf. At the same time, the chlorophyll content was determined by acetone process.

On the 0, 7^th, 14^th, 21^st, 28^th, 35^th and 42^nd d after heading, the fresh flag leaves of wild type and mutant were taken, and 0.2 g of each was put into the mortar, and 8 mL 0.1 mmol/L phosphate buffer (PB) and quartz sand were added to grind. Then put it into refrigerated centrifuge and centrifuged at 4 000 r/min for 10 min at 4℃, and the supernatant was used to measure the content of soluble protein. The Fv/Fm value was measured by Portable PAM Fluorometer PAM 2500 produced by WALZ company in Germany. The measurement method was referred to Genty et al. (1989).

3.3 Genetic analysis

F₁ was obtained by hybridization of es-h and wild type Hwacheongbyeo, es-h and Milyang23, respectively. And F₂ segregation population was obtained by inbreeding. The phenotypes of F₁ and F₂ were observed, and the segregation ratios of normal and premature plants in F₂ population were counted. The chi-square test under continuous correction was performed with Excel.

3.4 Fine mapping

DNA was extracted from the two parents and all individuals in the F₂ population and stored in an ultra-low temperature freezer. After investigating the phenotype, 5 normal plants and 5 mutant plants were selected from F₂ plants, and the DNA of each plant was mixed equally to construct two normal gene pools and mutant gene pools, respectively, which were used to screen the linkage markers of premature senility mutant genes. After determining the linkage markers, we continued to screen the linkage markers on the chromosome segment and used these markers to analyze the genetic population by PCR and electrophoresis and read the genotype data for the construction of linkage genetic map. The genetic map was constructed by genetic mapping software JoinMap 4.0. the recombination rate was transformed into genetic distance by Kosambi function, and the premature senescence mutant gene was located on the corresponding chromosome. According to the primary mapping, the genome sequences of indica rice 93-11 and japonica rice Nipponbare in this interval were downloaded from the Gramene website, and the insertion or deletion sites were searched through sequence alignment. The polymorphic markers were designed to analyze all the plants, and the target genes were fine mapped. In this study, a total of 2 079 F₂ and its derived F₃ plants were used to extract DNA, investigate premature senescence phenotypes, and fine mapping was performed by chromosome walking.

Authors’ contributions

MZM and BHJ were the experimental designers and executor of this study. JYM participated in data analysis and manuscript writing. WT, PRH, MY participated in the collection of some experimental data. JWZ and DXL were responsible for guiding the experimental design, data analysis and manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the National Key R&D Program of China (2017YFD0100504), Special Foundation for Provincial-School Co-construction Plan in Jilin Province (SXGJSF2017-6), and Project of Innovation and Entrepreneurship Training for College Students (201910183299).

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