4.1 Premature senescence and excess starch phenotype induced by LSES1 mutation
Assimilate partitioning has long been recognized as a target for crop improvement because it can limit the yield potential of crop plants. A phenotype involving an excessive accumulation of starch and growth retardation is often observed when any inhibition in the export of photoassimilate or in starch metabolism occurs in the leaves . In this study, the OsCKI1-deficient allelic mutant lses1 showed obvious premature senescence and an excess starch phenotype in leaves. Compared with WT, older leaf blades in lses1 displayed chlorosis at the tip from the third-leaf stage, and the chlorosis degree deepened with the development process and gradually developed to higher leaf positions (Fig. 1-A). In particular, the starch content and I2-KI staining analysis indicated that lses1 presented an abnormal starch accumulation phenotype in leaves during premature senescence (Fig. 1-B and Table S1). This phenotype was similar to that of three reported rice mutants, esl10 , ossac3 and ossac4 . Although LSES1 is allelic to OsCKI1/hbd2/LTRPK1/LTG1 and the mutation site in the lses1 mutant is similar to that in the ltg1 mutant (our unpublished data), no results have been reported on the premature senescence and excess starch phenotype in the leaves of OsCKI1-deficient allelic mutants except lses1. Therefore, the lses1 mutant is an ideal material for studying the molecular mechanism of premature leaf senescence and temporary starch metabolism via LSES1/OsCKI1 and the pleiotropism of LSES1/OsCKI1 in rice.
4.2 Global changes in the transcriptome and proteome in lses1
To comprehensively explore the mechanism of premature leaf senescence caused by temporary starch regulation and the changes in metabolic levels during senescence, transcriptome and proteome analyses were conducted, and the results showed global changes in mRNA and protein levels in lses1 leaves (Fig. 2-C and Fig. 3-B). Furthermore, subsequent functional analysis of the 4989 differentially expressed genes (DEGs) and 568 differentially expressed proteins (DEPs) showed that the DEGs were mainly involved in carbohydrate metabolic process, oxidation reduction and response to abiotic stimulus (Fig. 2-D), while the DEPs were heavily involved in translation, cytoplasmic part and biosynthetic process (Fig. 3-D). Gene expression is usually affected by transcription, posttranscriptional regulation, RNA splicing, translation, and posttranslational modification, which results in changes in mRNA and protein levels that are not always the same. In this study, approximately 20% of the identified proteins were also found to be regulated at the transcriptional level. The congruence between proteomics and transcriptome data seems to be very poor, which is consistent with general observations [4, 5, 18, 24, 25].
4.3 Metabolic imbalance induced an excessive accumulation of starch and affected glycolysis/TCA cycle in lses1 leaves
The metabolism of temporary starch may be a complex network formed by the coordinated expression of numerous genes. In this study, several key proteins involved in starch biosynthesis, such as AGP-L (glucose-1-phosphate adenylyltransferase large subunit), GBSSII (granule-bound starch synthase II), SSS1 (chloroplastic soluble starch synthase 1) and SSI (chloroplastic starch synthase I), all exhibited increased abundance in lses1 (Fig. 5). Notably, AGP-L is the first rate-limiting enzyme in starch biosynthesis, and its overexpression rice plants show significantly increased starch content in leaves . Moreover, several β-amylases in lses1 showed down-regulated transcription levels. A striking sample is BAM3 (Fig. 5), which encodes the most important β-amylase that strongly participates in the decomposition of temporary starch in leaves at night, and its functional deletion mutant exhibits a higher transitory starch accumulation phenotype in leaves than does other β-amylase deletion mutants . Excessive accumulation of leaf starch affects the normal development of plants, resulting in plant growth retardation, decreased yield, and even premature senescence, which has been reported in typical model plants, such as rice and Arabidopsis [21, 28, 29].
Furthermore, the expression levels of multiple genes/proteins related to carbohydrate metabolism downstream of starch metabolism in lses1 were almost all upregulated (Fig. 5). SUS (sucrose synthase 1) overexpression provides more UDP-glucose and fructose for various metabolic pathways , while FRK (fructokinase-2) upregulation catalyses the formation of more fructose-6-phosphate derived from fructose and channels sucrose into the glycolytic pathway . It could be speculated that the substrate of glycolysis is more biased towards fructose and sucrose rather than glucose derived from starch due to the accumulation of starch in the chloroplast, which reduces the transport of maltose and glucose to the cytoplasm. The increased intermediate products further induce the upregulation of downstream glycolysis/TCA cycle-related genes (Fig. 5). On the other hand, the additional sucrose consumption seemed to induce the upregulation of MEX1 (chloroplastic maltose excess protein 1-like) and TRE (trehalase) to maintain a stable sucrose content (Fig. 5). MEX1 is the main carrier of maltose from chloroplasts to the cytoplasm for sucrose synthesis , while TRE is involved in trehalose metabolism, playing an important role in regulating the balance of sucrose and trehalose contents .
Taken together, these results implied that the metabolic imbalance of transitory starch, which appeared as increased starch biosynthesis and incomplete starch degradation, induced the excessive accumulation of starch in lses1 leaves, which coincided with the abovementioned phenotype and physiological characteristics in lses1.
4.4 Increased premature senescence-related metabolism in lses1
Comparative transcriptomic and proteomic analyses implied that in addition to carbohydrate metabolism-related genes/proteins, other mechanisms might contribute to explaining the premature senescence phenotype of lses1 mutant leaves. The leaves of lses1 presented increased ROS and MDA contents compared to WT (Fig. 1-C top). These physiological differences were also reflected in the changes in mRNA and protein levels. For example, the protein psaC (photosystem I iron-sulfur centre) and gene CR7 (cytochrome bc1 complex subunit, complex III) in lses1 showed increased protein and transcript abundance, respectively (Fig. 5). Among them, psaC is involved in ROS generation in the chloroplastic electron transport chain , while CR7 affects the electron transfer rate of the mitochondrial respiratory chain . The leaves of lses1 showed lower SOD, CAT and POD activities compared with WT (Fig. 1-C bottom-left). Interestingly, several L-ascorbate peroxidases, such as APX1 and APX3, in lses1 all exhibited increased transcripts and decreased protein abundance (Fig. 5), which suggested that the expression of APXs is posttranscriptionally regulated . In addition, the contents of chlorophyll a, chlorophyll b and total photosynthetic pigment in lses1 leaves were significantly higher than those in WT (Fig. 1-C bottom-right). As expected, several photosynthetic pigment anabolism-associated genes in lses1 were regulated. For example, CAO (chlorophyllide a oxygenase), which is involved in chlorophyll b biosynthesis , and rccR (putative red chlorophyll catabolite reductase), which is involved in chlorophyll a degradation , showed down- and upregulated transcription levels, respectively (Fig. 5). Notably, SGR in lses1 exhibited reduced transcription (Fig. 5); this gene triggers chlorophyll degradation in natural or dark-induced leaf senescence , and both its deletion and overexpression will affect the degradation of chlorophyll in rice leaves .
In brief, the results showed increased premature senescence-related metabolism in lses1 and suggested consistency between the transcriptomic and proteomic analyses and the physiological determination of lipid peroxidation and ROS-scavenging enzymes.
4.5 Increased nucleotide degradation and inhibited cell proliferation in lses1
Senescence is considered a self-saving strategy in plants, during which plants recycle and deliver nutrients from degraded proteins, lipids, and nucleic acids to still growing sites . A similar senescence-induced plant adaptation also occurred in the lses1 mutant described herein. In this study, APRT (adenine phosphoribosyltransferase form 2) in lses1 showed a higher protein abundance level than that in WT (Fig. 5); this gene is involved in step 1 of the subpathway in the salvage pathway that synthesizes AMP (adenosine monophosphate) from degraded adenine . Interestingly, TET6 (tetraspanin-6), which is considered a senescence-associated protein and is involved in leaf and root growth via negative regulation of cell proliferation in Arabidopsis , was found to be expressed in lses1 but not in WT (Fig. 5). Additionally, KRP6 (cyclin-dependent kinase inhibitor 6) was found to be overexpressed in lses1 mutant (Fig. 5), which accumulates in Arabidopsis lacking CK1s/AELs will inhibit cell proliferation . Taken together, these results implied that increased RNA/DNA degradation (a trait of PCD), nutrient recycling of nucleotides and inhibited cell proliferation occurred in the lses1 mutant during senescence.
4.6 ABA may involve in multiple metabolic regulation via LSES1/OsCKI1
The rapid accumulation of ABA is considered one of the key characteristics of plants in response to abiotic stress , and it is related to the changes in starch metabolism induced by stress . In this study, four proteins associated with ABA accumulation and signaling, including ASR5 (abscisic stress-ripening protein 5), ASR2 (abscisic stress-ripening protein 2 fragment), PYL5 (ABA receptor 5) and SAPK (serine/threonine-protein kinase), showed significantly increased abundances in the lses1 mutant (Fig. 5). Among them, ASR5 is involved in the ABA-mediated stomatal closure pathway in response to drought and osmotic stress, and rice plants overexpressing ASR5 gene exhibit higher endogenous ABA accumulation . PYL5 functions as a positive regulator of abiotic stress-responsive gene expression , which together with SAPK2, is part of an ABA signaling unit that modulates seed germination and early seedling growth . OsCKI1/hbd2/LTRPK1/LTG1/EL1, which is involved in hybrid breakdown, root development, hormone response, cold adaptation and heading, has been cloned utilizing multiple rice mutants with different phenotypes [7-11]. Notably, Liu et al. (2003) showed that OsCKI1 may be involved in the regulation of gene expression and mediating the interaction of ABA and other hormones signaling through hierarchical phosphorylation . Recent study reports that mutation of the CK1s causes reduced phosphorylation, ubiquitination, and degradation of ABA receptors PYR/PYLs, thereby enhancing the ABA responses , which is similar to the result observed in the lses1 mutant. In addition, transcriptional and translational control seems to play important roles in the regulation of starch metabolism in response to stress. Multiple primary carbohydrate metabolism-related genes were found to be regulated by ABA in previous transcriptomic and proteomic studies [51-53]. For example, AGP-L (glucose-1-phosphate adenylyltransferase large subunit) which encodes the large subunits of AGPase involving in starch biosynthesis showed up-regulated expression in lses1 mutants (Fig. 5). The accumulation of AGP-Ls transcript is known to cooperatively regulated by ABA and sucrose, and their expression patterns were elevated significantly by the co-treatment of sucrose and ABA [54, 55]. Taken together, these results implied that LSES1/OsCKI1 mutation might enhance the ABA response, and then the increased ABA cooperated with sucrose to regulate downstream starch metabolism-related gene expression, which ultimately leads to excessive starch and premature senescence phenotypes in the leaves of lses1 mutant.
4.7 The PPI network may affect the development and metabolism of leaves in lses1
Analysing protein-protein interaction (PPI) networks has become increasingly important for further exploring the biological characteristics of proteins during plant development. In this study, 568 DEPs were used to form a PPI network, and four highly related functional clusters were found in this interactome by the MCODE  Cytoscape plugin (Fig. 4-A). In cluster 1, four proteins, A2XZF9, B8AZD7, B8ACZ5 and B8AE20, encoding the eukaryotic translation initiation factor 3 subunit, were found to interact, which may be due to their functional links in modulating the global translation rate to regulate cell growth, proliferation and differentiation . These four proteins all showed upregulation in lses1 (Table S4). In cluster 2, B8AGW7 (acetyltransferase component of pyruvate dehydrogenase complex) interacted with A2XTX6 (3-oxoacyl-[acyl-carrier-protein] synthase). The pyruvate dehydrogenase complex synthesizes acetyl-CoA, which binds to acyl carrier proteins to participate in fatty acid biosynthesis . Both B8AGW7 and A2XTX6 also showed upregulation in lses1 (Table S4). In cluster 3, two carbohydrate metabolism-related proteins were found to interact: A2WLL3 (ATP-dependent 6-phosphofructokinase) and A2YKG1 (glucose-6-phosphate 1-dehydrogenase). Their interaction is due to the connection of substrates between glycolysis and the pentose phosphate pathway. Both A2YKG1 and A2WLL3 were upregulated in lses1 (Table S4). In cluster 4, two proteins related to phospholipid metabolism were found to interact: B8AL07 (phosphatidylserine decarboxylase proenzyme 1) and Q9LKM2 (phospholipase D). Their interaction also includes N-acylphosphatidylethanolamines (NAPEs), which exist during cell damage . Phosphatidylserine decarboxylase proenzyme 1 and phospholipase D are involved in the synthesis and decomposition of NAPEs, respectively [59, 60]. Interestingly, Q9LKM2 was downregulated but B8AL07 was upregulated in lses1 (Table S4), which suggested that these two proteins may have an antagonistic effect. Taken together, these results implied that the PPI network might play an accessory role in promoting the apoptosis-related metabolism level of lses1 leaves.