The interplay of carbon and nitrogen distribution: Prospects for improved crop yields

Growth and productivity of plants primarily depend on the balanced distribution of carbon (C) and nitrogen (N) among different organs. Previous studies on crop improvement have focussed on the C or N assimilation and distribution. However, recent findings reveal that C and N form a complex integrated network and are often dependent on each other to affect crop productivity. The underlying physiological and molecular mechanisms involved in the coordinated distribution of C and N among different plant organs are yet to be fully uncovered. Crucial roles in regulating C and N balance are played by transporters that mediate their movement across different organs. In Cotton, which has an indeterminate growth pattern, source–sink assimilate distribution could be a major bottleneck impeding fibre productivity. This review summarises our current understanding of C and N transport mechanisms, explores and compares different physiological and molecular approaches involved in the C–N distribution cascade, including cotton and other plant species. A comprehensive understanding of these integrated regulatory mechanisms is crucial for improving crop yields and fibre productivity.


INTRODUCTION
Plants rely on assimilated carbon (C) and nitrogen (N) for optimal growth and development. 1 Hence C and N are strongly intertwined. In plants, C is assimilated in the form of sucrose, which is a transportable form of C, exported from the source leaves (vegetative plant part) to the sink organ (reproductive tissues) via phloem. Meanwhile, N assimilated in the form of inorganic nitrogen compounds, such as nitrate and ammonium, are converted to amino acids in the shoot or root. 2, 3 The process of N assimilation is illustrated in Figure 1. The synthesised amino acids are transiently stored in the source leaves and then relocated to the sink organ. 4,5 Sink organs, such as flowers, fruits, seeds, and younger leaves, are known as the primary reserve for C and N assimilates, while source organs, such as older leaves and green plant parts, produce and export photoassimilates through photosynthesis. However, it is worth noting that not source organs to overall productivity can vary depending on the plant species and environmental conditions.
Approximately three-quarters of the assimilated carbon and amino acids produced in source leaves are transported to the sink organs based on the sink's metabolic need. 6 Once translocated, the assimilates (sucrose and amino acids) are distributed and utilised for plant growth and development. 6 Numerous molecular and biochemical studies have highlighted the importance of sucrose and amino acid partitioning in plant growth and development. [7][8][9] These studies have paved the way for manipulating vascular tissues or pathways involved in sucrose and amino acid transport to improve crop yields. Yadav et al. 10 demonstrated that manipulating the expression of genes encoding sucrose or amino acid transporters (AATs) led to preferential accumulation of assimilates in targeted plant organs, thus promoting sink growth. Hence, gaining a more comprehensive understanding of the physiological and molecular mechanisms involved in the coordination between C and N metabolisms could be leveraged to maximise crop yields.
Cotton (Gossypium hirsutum L.) is a significant cash crops grown worldwide for cottonseed oil and fibre production. The top cotton-producing countries F I G U R E 1 Schematic representation of coordination between carbon and nitrogen assimilation in plants. Root uptake and transport of inorganic N (NO 3 − and NH 4 + ) is regulated by nitrate (NRT) and ammonium (AMT) transporters via the xylem (1). NO 3 − and NH 4 + are subsequently transformed into amino acids via the action of a series of N assimilation enzymes: nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and asparagine synthetase (AS) (1) and (2). Similarly, assimilated carbon during photosynthesis is converted to 2-oxoglutarate (produced from isocitrate via the TCA cycle) to further synthesise glutamate needed for amino acid synthesis (3). 58 -MODERN AGRICULTURE are China (5879 metric tons, MT), India (5334 MT), Pakistan (1306 MT), the United States (3815 MT), and Brazil (2678 MT). 11 Cotton fibres are a vital raw material for the textile industry, making the need to improve fibre yield and productivity urgent. Cotton exhibits indeterminate growth habits with its vegetative (source) and fruiting branches (sink) competing for photoassimilates. Understanding the source-sink relationship in cotton development is therefore critical. Several studies on enhancing crop yield are majorly funnelled towards C or N metabolism and their distribution processes. [12][13][14][15][16] In this review, we summarise our current understanding of C and N transport mechanisms and their distribution processes. We explore different aspects of physiological and molecular processes underlying the C-N cascade in relation to crop yield, comparing among different plant species, including cotton. We also review the omics techniques, which can potentially identify the key regulators involved in C and N interplay, as well as their application in enhancing C-N balance and yield. Manipulating sucrose and amino acid transport in plants through transgenic techniques can serve as a precursor to enhancing assimilate distribution necessary for sink growth. Elucidating these integrated mechanisms involved in C and N distribution processes would help improve crop growth and productivity.

THE TRANSPORT MECHANISM OF CARBON ASSIMILATES FROM SOURCE TO SINK ORGANS
Sucrose is the predominant form of C synthesised in the cytoplasm. 10 It can either remain in the cytoplasm for other cellular functions or be compartmentalised within the vacuole. After compartmentalisation, sucrose is transported from the source to the sink organs on demand, while the remaining sucrose is converted into starch and utilised in the chloroplast. 17 The amount of sucrose available for export is dependent on various factors, including but not limit to the efficiency of carbon fixation, light reaction, and the amount of exported triosephosphate. 18 These factors are crucial in maintaining enhanced source-sink strength since alterations in any of these factors can significantly affect photoassimilate transport, thereby disrupting the crop yield potential.
The mechanisms involved in active phloem loading have gained particular attention in past few years. To date, three putative active phloem loading mechanisms have been discovered in plants. 19 The first is the symplastic loading, which facilitates the movement of synthesised sucrose from mesophyll cells to sieve elements via plasmodesmata (PD), acting as a bridge across the cell wall ( Figure 2). The second is an apoplastic mechanism that involves the transfer of sucrose from mesophyll to companion cells across the concentration gradient ( Figure 2). The third mechanism is polymer trapping, which involves the conversion of sucrose into relatively large sugar polymers, including verbacose, raffinose, and starchyose, that are supplied symplastically via intermediate cells. 20,21 The active phloem loading into the apoplast is facilitated by sucrose transporters (SUTs or SUCs) and the recently identified proteins, Sugars Will Eventually be Exported Transporters (SWEETs). 22,23 SWEET proteins mediate sucrose retrieval and transport through the phloem, where sucrose is imported into the sieve elements or companion cells by proton symporters. 20 The regulation of sucrose loading pathways remains inadequately explored. At the cellular level, other sugar-specific transporters also facilitate intercellular sucrose transport and modulate sugar fluxes. 24,25 Previous studies show the crucial role of sucrose transporters in crop yield improvement; for example, impaired function of SUT1 reduces fresh weight in early developmental stages in potato, 26 and lack of OsSUT2 function limits sucrose import to sink organs, causing physiological damage and yield loss in rice (Oryza sativa L.). 27 Thus, effective loading and unloading of sucrose by transporters directly affects crop productivity.
Sucrose transporters are located within the phloem, including collections phloem, transport phloem, and release phloem. 18 The long-distance transport of sucrose starts from the collection phloem in the source, through the transport phloem, to the release phloem for final delivery to the sink tissue. 28 In the sink organ, sucrose molecules primarily follow the symplastic route for unloading in the sink tissue, but the apoplastic route is required for effective transport if the symplastic pathways are disrupted 29 (Figure 2). The mode of sucrose unloading at the sink organ depends on the sink strength and developmental stage of the plant. 30,31 For instance, in cotton, 32 potato, 33 apple, 34 and jujube, 31 sucrose unloading switches from the apoplastic pathway to the symplastic one on a developmental stage-dependent manner. In contrast, unloading sucrose in grapefruit appears to be symplastic at the early and mid-developmental stages, whereas it switches to the apoplastic mode during the later developmental stages. 35 The sucrose unloaded at the sink organ can be broken down into fructose and glucose by invertase (INV) 36 or converted to fructose and uridine diphosphoglucose (UDPG) by sucrose synthase (SUS). Albeit the energy demand for the degradation is usually higher for INV than for SUS. 21,37 INV is a key enzyme in apoplastic sucrose unloading. 38 CWIN stimulates cell division in the sink organ during the seedling stage, thereby enhancing sucrose unloading by converting sucrose into hexose. 37,39 Therefore, enhancing the endogenous CWIN activity is a promising approach to mitigate the effect of sink abortion, which has significant negative impacts on crop yields.

NITROGEN ASSIMILATION AND PARTITIONING PROCESSES
The assimilation, transport, and partitioning of N among plant organs are essential steps for maintaining plant cellular structures and functions, ultimately leading to improved plant growth. 40,41 Plants generally take up inorganic N and assimilate it in the form of amino acids  through N-assimilatory enzymes, such as nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS). [2][3][4] The process of N assimilation is depicted in Figure 1. After conversion, these amino acids are utilised for various metabolic processes in the roots, but a substantial amount is also transported via the xylem to the vegetative parts (source leaves). Source leaves use the amino acids for metabolism and protein synthesis, and the remaining amino acids are loaded into the leaf phloem where it is reallocated to different storage organs (sink organs), including young leaves, roots, and developing seeds. 42,43 Therefore, amino acids are primarily considered the transportable form of N. 42 The AAT family has been identified in several crops. [44][45][46] However, due to the structural complexity of cotton, little is known about the AAT gene family in cotton. Yang et al. 47 recently identified and investigated the expression profile of the GhAAT gene during fibre development. Eight genes were found to be specifically expressed during fibre elongation and maturity. 47 This finding laid the ground for the functional characterisation of the AAT gene family in cotton. The complexity of source-sink interaction presents potential bottlenecks to long-distance N transport with amino acid loading and unloading in the phloem being particularly important. Zhang et al. 42 investigated this issue by manipulating two transport steps: amino acid loading and unloading at the sink cell through the editing of the pea amino acid permease (PsAAP1) in the pea genome. The increased expression of PsAAP1 transporter in the phloem, irrespective of N supply, led to enhanced phloem loading, long-distance N transport, protein levels, and yield. 42,48 Comparable results were obtained by Carter and Tegeder 49 using ureides as a long-distance form of N in soybean (Glycine max). Furthermore, the increased expression of ureide permeases (UPS1) in the vascular tissue improved Nbased nutrition and seed development by facilitating the reallocation of N from the source to the sink cells. 49 Taken together, it can be inferred that an increased expression of amino acid (and ureide) transporters facilitates positive feedback in N acquisition, amino acid synthesis, and its transport, thereby improving sink strength.
Interestingly, amino acid loading and transport not only affect sink growth but also influence source capacity. 42 These processes are also facilitated by the activities of AATs. 4,50 For example, increased source capacity and seed protein levels were observed in pea (Pisum sativum) overexpressing PsAAP1 under high N conditions, indicating the potential role of PsAAP1 in regulating assimilated carbon and amino acids. 42 Subsequent findings demonstrated that the regulation of amino acids allocated at the source leaves affects photosynthesis and that efficient coordination between F I G U R E 2 Schematic illustration of the transport steps involved in the active phloem loading and unloading. After synthesis, sucrose (blue dots) moves from mesophyll to companion cells and finally to the collection phloem via symplastic and apoplastic pathways (1). On arrival at the collection phloem, sucrose is imported into the transport phloem through a high concentration gradient (2) and then to the release phloem (3), where it is unloaded at the sink organ by sucrose transporters (SUTs) (4). Sucrose is unloaded through either symplastic or apoplastic paths depending on the plant species and its developmental stages (5). Cotton is used as the model plant for illustration.

60
-MODERN AGRICULTURE carbon and nitrogen distribution processes is critical for seed performance regardless of the N supply. 51 Further, transgenics-based experiments showed that manipulating long-distance amino acid transport primarily affects C and N metabolism. 10,52 However, less is known about their complex interactions with other nutrients. Understanding this integrated cascade is crucial for future research on how C, N, and mineral nutrients are simultaneously regulated to attain the much-expected yield prospect. Nonetheless, integrating the C-N-nutrient cascade requires understanding the effects of different N status on carbon partitioning processes as summarised in the following section.

Effects of nitrogen status on carbon and nitrogen partitioning and their impact on crop growth
Most of the changes in plant biomass can be attributed to alterations in C and N partitioning processes that are linked with N status in plants. 53,54 N-deficient leaves often exhibit reduced chlorophyll synthesis, lower levels of soluble proteins and amino acids, and retarded growth ( Figure 3). However, under such conditions, a substantial amount of free amino acids is observed in the sink cells, such as roots, fruits, seeds, flowers, and younger leaves, indicating that N assimilation in roots is inadequate to meet the N metabolic requirements in leaves ( Figure 3). 55 An increase in free amino acids and proteins has been observed in cotton leaves with increasing N supply, 56 consistent with findings in Leymus chinensis. 53 In N-sufficient conditions, L. chinensis showed a sequential increase in the free amino acid content in the leaf, stem, and roots, until further N increase led to a slight decline. 53 This decline was due to the accumulation of soluble proteins and amino acids, which exceeded the quantity required for plant growth. 57 Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) constitutes approximately 30%-50% of the leaf-soluble protein, so reductions in soluble proteins typically translate into the loss of Rubisco. 58 Therefore, it becomes essential to determine Rubisco activity during photosynthesis to improve crop yields. 59 The influence of N status goes beyond its effect on N metabolism and involves the regulation of nonstructural carbohydrate partitioning. 53 In cotton plants, studies have shown that N deficiency leads to the accumulation of more starch and sucrose in the leaves than in N-sufficient plants ( Figure 3). 60 This phenomenon was also observed in a previous study of olive leaves under low N-conditions. 55 The accumulation of starch and sucrose could result from a drastic reduction in the amount of sucrose synthesised and the export rate to the sink. 60 As a result, the import of plant carbohydrates to the sink was hampered, leading to the reduced boll size, fibre length and strength, and seed biomass ( Figure 3). 60 Increasing N supply led to a sequential reduction in sucrose accumulation in cotton leaves compared to other N regimes. 60 These findings were contradictory to the previous findings showing the increased leaf sucrose content. 61 Similarly, recent research on L. chinensis affirmed a drastic reduction in total leaf soluble sugar, sucrose, and starch contents with a progressive increase in external N supply (N was supplied in form of NH 4 NO 3 ) consistent with Tang et al. 60 and Gong et al. 53 (Figure 3). The increased N nutrition may strengthen the sink and expedite its demand for resources (nutrients and energy), thus allowing efficient redistribution of resources from the source leaves. 62 Triose phosphate utilization rate (VTPU) facilitates sucrose synthesis and mediates the conversion of C to starch or sucrose. 63 In L. chinensis, the increase in VTPU was proportional to N supply until further increase resulted in a drastic reduction. Furthermore, increasing VTPU led to a gradual increase in the sucrose content in the stem but a decrease in the leaf, suggesting that N nutrition enhances VTPU-mediated sugar import to the sink organ rather than accumulating it in the source leaves. All these processes encourage ATP synthesis and photosynthetic rate. 64 An additional supply of nitrogen enhances C and N metabolism and partitioning processes, leading to improved crop yield. These studies highlight the interlinked relationship between C and N metabolism in plants.

ENVIRONMENTAL CUES AFFECTING THE SOURCE-SINK RELATIONSHIP AND PLANT GROWTH
Plants are continuously exposed to environmental changes, and as such, they develop adaptive mechanisms to survive. Abiotic stresses, such as drought, light stress, and elevated CO 2 , are the major limiting factors that affect cotton yield and productivity. The effects of drought, light stress, and elevated CO 2 on the source-sink relationship and plant development are discussed in this section.

Drought stress
Drought stress is a severe limiting factor that causes physiological damage and growth retardation in plants. Drought stress alters the balance of source-to-sink relation and triggers the accumulation of more sucrose at the source leaves, reduces free amino acids and soluble proteins, and disrupts assimilate transport to the fruiting branches, where growth occurs. [65][66][67] Drought stress represses the accumulation of sucrose and starch in sink cells by reducing the activity of sugarmetabolising enzymes, including sucrose phosphate synthase (SPS), soluble acid invertase (SAI), and SUS. 68 In drought stress-treated cotton plants, reduction in photosynthetic efficiency and fibre retardation are observed. 69 The deposition of sugar at the source leaves could compensate for the drop in free amino acids and soluble proteins in response to drought stress, which sustains cell turgor in leaves. 66 This MODERN AGRICULTURE phenomenon further supports the C and N coordination in response to drought.
Sucrose transporters, namely SWEETs and SUTs, play a key role in regulating the transport of assimilates from source-to-sink under drought stress. In Arabidopsis thaliana, AtSWEET11, AtSWEET12, and AtSUC2 are known to mediate sucrose transport to the root in response to drought. 70 Recent research has shown that the increased expression of sucrose transporter genes GhSUT4, GhSUT9, and GhSWEET55 in cotton leaves enhances sucrose transport in response to short-term drought. 71 However, under prolonged drought conditions, the upregulation of these genes was drastically reversed. A similar trend is observed in soybean, where the initial upregulation of sucrose transporter genes GmSUC2, GmSWEET6, and GmSWEET15 was preceded by a drastic decline in gene expression under prolonged drought conditions, impeding sucrose export to the sink organ. 72 This disruption of sucrose export under long-term drought stress could be attributed to drought-induced photosynthetic inhibition, phloem blockage, and lower sink demand. 72 Drought stress reprograms N-responsive gene expression, reduces NO 3 − uptake in roots, and increases N deposition in shoots in cotton plants. 71 Similarly, drought negates root NO 3 − uptake in Populus simonii, 73 barley, 74 and Malus prunifolia. 75 Under prolonged drought stress, GhNPF4.6 was downregulated in the cotton root, suggesting the low N demand during water-deficit conditions. Meanwhile, short-term drought treatment led to the increased expression of GhNPF4.6 F I G U R E 3 The diagram illustrates the feedback consequences of N status on C and N distribution among plant organs. Cotton was used as a model plant for illustration and sectioned as N-sufficient and N-deficient plants. Under the N-sufficient condition, the root assimilates adequate NH 4 + required to meet leaf N demand (more NH 4 + ) (1). Then, NH 4 + is assimilated into amino acids (AA) in the source leaves (2) and subsequently distributed by transporters (3) among plant organs for improved source and sink growth (4). The synthesised amino acid at the source leaf supports photosynthetic carbon assimilation, thus improving sucrose synthesis. The synthesised sucrose is relocated from the source leaf (2) to the sink organ based on the demand (as indicated by the black arrow), leaving lesser sucrose at the source leaves and more sucrose deposits at the sink organs (cotton, fibre, flowers, and bolls) (4). Conversely, N-deficient plants (on the right side) have more sucrose and fewer amino acids in the source leaves (A) since the root cannot assimilate an adequate amount of NH 4 + needed to meet leaf N demand (B). Under this condition, lesser sucrose is allocated to the sink organ due to the hampered relocation of sucrose to the sink organ (C). However, the genes responsible for such inhibition are unknown as indicated with a question mark '?' on the N-deficient side.

62
-MODERN AGRICULTURE due to the excessive sugar-induced N accumulation in cotton, 71 indicating a more intense feedback effect of drought on the cotton biomass than N uptake. 76

Effect of light
Plants are highly responsive to light conditions, particularly to low light or shaded environment, which can significantly impact crop performance. 77 In the absence of light, seedlings depend on their C assimilate reserves for hypocotyl elongation, resulting in shoot-over root growth and inhibition of root growth. As a result, the shoot apex is directed towards the light. 78 Upon exposure to light, de-etiolated seedling starts photosynthesis, which leads to the production of sucrose that is transported to the roots. 79 To adapt to low light conditions, plants employ a mechanism similar to the battery system. During the day, a fraction of the assimilate produced through photosynthesis is stored as starch, while at night, when photosynthesis is not active, the stored assimilates are mobilised to provide energy, which are essential for sustaining plant growth. 80 Thus, starch availability during periods of darkness and the length of photoperiod are principal determinants of plant growth. If the period of darkness is prolonged, catabolism is activated and starch and sucrose reserves become depleted, leading to stunted growth. 81 Therefore, proper coordination between starch degradation and growth regulation becomes imperative to avoid excessive sugar deposition or depletion during periods of catabolism. This coordination can be achieved through two mechanisms: (i) direct reprograming of the growth rate in plants by the circadian clock and (ii) indirect control of the rate of starch degradation and the quantity of sucrose available for growth by the circadian clock. 80 Unlike short-day plants that exhibit growth retardation at dawn, long-day plants show improved crop growth during the day and at night. 82 N assimilation is closely linked to photorespiration, photosynthesis, and the TCA cycle. This coordination is essential for maintaining the output of photoassimilates required for plant development under fluctuating light conditions. 77 Leaves that are directly exposed to sunlight require higher levels of N to support efficient carboxylation compared to the shaded leaves. 83 The increased leaf N content indicates enhanced N metabolism under weak light conditions. Since N assimilation requires carbon for growth support, amino acids and other N reserves can also compensate for C deficiency to improve plant tolerance to low light conditions. 84

Higher carbon levels affects nitrogen metabolism and overall crop yield
The global carbon dioxide (CO 2 ) level is predicted to exceed 700 μmol/mol by the year 2050, rising faster than expected. 85 Investigating the feedback effect on N metabolism is crucial for improving crop yields. Although increased CO 2 levels significantly improve plant biomass under C-limited conditions, most plants exposed to elevated CO 2 (EC) fail to reach their full growth potential due to nitrogen availability constraints. 86 Though the impact of EC on N metabolism has been widely studied in several crops, 87,88 there is no recent literature available on the detailed impact of EC on N metabolism in cotton plants. One of the most intriguing features of plants exposed to high CO 2 is reduced plant N and protein content. 89,90 Furthermore, studies have shown that exposure to EC with optimal N supply can lead to a one-tenth reduction in the grain protein content without affecting or reducing crop yield. 91 However, N concentration in seedlings is typically reduced under EC, which limits plant growth. This reduction is linked to the accumulation of non-structural carbohydrates, reduction in N assimilation, and changes in N signaling. 88,92,93 The reduction in N content also contributes to the significant loss in Rubisco activity, 94 and the suppression of photorespiration under EC leads to further decreases in N content. 95 Moreover, EC negatively impacts N nutrition by disrupting the export of photoassimilates from the source leaves and reduces sink strength. 96 Dong et al. 97 investigated the possibility of mitigating N reduction induced by EC/N deficiency through external supply of N (NO 3 − form). Their study found that the addition of NO 3 − significantly increased N assimilation and content in seedlings, indicating that high NO 3 − levels positively regulate N assimilation under EC conditions. Therefore, increasing the application of NO 3 − fertilizer to counteract the negative effects of EC/ N deficient conditions is a practical approach.
Studies have also shown that EC regulates the activities of genes involved in N assimilation, including GS, NR, and glutamate synthase (GOGAT) isoforms. 88 In wheat, EC exposure led to the upregulation of NR activity in the root. 88 Furthermore, EC led to enhanced amino acids but negatively regulated soluble proteins in the leaves, 98 suggesting alterations in leaf GS/GOGAT activity. While GOGAT activity was reduced in the leaves, it was increased in the root under EC exposure, indicating a shift in N assimilation towards the roots. 88 These observations are consistent with previous findings, proposing that EC enhances root nitrate assimilation. 98 Overall, alterations in genes regulating N assimilation under EC exposure aim to maintain efficient C and N distribution among plant organs. 99,100 Additionally, EC also regulates N-signalling genes under different N levels. The expression of N-signalling genes is increased under NO 3 − limiting conditions, but decreased under sufficient N with EC treatment. 88 Thus, the expression of these N-signalling genes is critical for C and N distribution among plant tissues and may exert a feedback effect on plants' tolerance to N deprivation.

REPROGRAMING C AND N DISTRIBUTION FOR IMPROVED CROP YIELDS
Several factors are reported to affect the phloem transport of C and N and their distribution; however, manipulating the activity of sugar and N-related MODERN AGRICULTURE -63 transporters could be a more pragmatic way to improve phloem assimilate transport, sink strength, and plant growth.

Contributions of sucrose transporter to carbon and nitrogen distribution
Sink growth are highly dependent on photoassimilates imported by the phloem. 101,102 However, inefficient sucrose export from the leaves via SUT phloem loaders can adversely affect photo assimilate import. 103,104 Thus, manipulating the activity of SUT transporters is a promising strategy for increasing the phloem transport of C photoassimilate (refer to Table 1 for further details). For example, the overexpression of phloemspecific SUC2 in rice led to enhanced C import to the sink, thereby improving grain (sink) size and yield. 110 In contrast, the overexpression of SUT1 in the phloem caused growth retardation in Arabidopsis, probably due to disruptions in the carbon to phosphorus balance. 114 Lu et al. 52 showed that C:P imbalance could be avoided by increasing sucrose import to the seeds using a 'pull' approach. The differences in the outcomes of rice and Arabidopsis are attributed to the use of different promoter constructs in developing the transgenic lines, which caused the variations in specific sucrose transporter functions and signalling processes. 103,115 Nonetheless, SUT1 overexpression in the phloem and embryo alleviated potential feedback inhibition of sucrose transport to the growing sink organ in Arabidopsis. 114 Furthermore, the overexpression of SUT1 also enhanced sucrose transport from source leaves to the sink organ, thereby increasing seed yield in pea. 52 It is worth noting that efficient sucrose export from the leaves not only improves sink strength and growth, but also mediates photosynthetic rates. 116 The activity of the sucrose exporter is expedient as excessive leaf sugar accumulation is known to lower the photosynthetic rate, 117 and the efficient sucrose transport helps resolve this complexity. In Arabidopsis, mutation of AtSWEET11 and 12 genes resulted in excessive accumulation of starch in the leaves, reducing photosynthetic efficiency, 118 highlighting the involvement of AtSWEET11 and 12 in leaf sucrose export. Similar findings were obtained in maize (Zea mays L.), where photosynthesis was found to be inhibited in a triple knockout mutant, zmsweet13 a,b,c. 112 The overexpression of AtSWEET1 homologue, CST1 (Closed stomata1), improved photosynthesis by regulating the stomatal movement and sugar content in maize. 119 In jujube (Ziziphus zizyphus), the overexpression of ZjSWEET2.2 resulted in enhanced phloem loading, thereby reducing the leaf carbohydrate content in the mesophyll cells. 111 The overexpression of SUT1 in pea markedly increased the photosynthetic rate, gene expression related to C-fixation, sucrose synthesis, transport, and efflux, 52 validating the hypothesis that the sucrose export process in SUT1-OE leaves increases carbohydrate fixation, synthesis, and partitioning. This phenomenon also ascertains the outcomes of expression studies in rice phloem overexpressing SUC2. 110 The importance of ZmSUT1 in sucrose distribution has been widely discussed in maize. 117 As such, Ding et al. 113 investigated the impact of ZmSUT1 expression on sucrose distribution in cotton, where it was expressed in cotton under the control of a senescence-inducible PSAG12 promoter and a seed coat-specific BAN promoter. Among these, BAN::ZmSUT1 exhibited an increased photosynthetic rate and improved sugar deposition in the boll and fibre, highlighting the potential roles of ZmSUT1 in promoting fibre formation by reprogramming sugar partitioning. 113 To further investigate how assimilate partitioning impacts fibre growth and seed yield, nine SUT gene family members were identified in cotton, which are critical for assimilate partitioning. 32 Among the identified members, GhSUT1-L2, expressed in mature leaves, was shown to promote the accumulation of carbohydrates in the leaves and its silencing disrupted phloem transport. Elucidating the mechanisms underlying phloem loading in cotton is a promising strategy to improve assimilate partitioning to the sink organs (bolls, flowers, and fibres). These findings suggest that one possible way to attain a high photosynthetic rate is to enhance the downstream transport capacity for withstanding the flow of deposited C and the sink's capacity to utilise it.
At the sink level, the uptake capacity of sucrose during the transition stage of seed development in pea was improved by the seed-specific overexpression of SUT. 120 Despite the increase in the seed growth rate, starch amount and dry seed weight were unaffected in the transgenic plants. 52 Also, sucrose levels were enhanced in the dry seed during embryo development, while an increase was observed in the starch levels beyond the storage phase. 52 These findings demonstrate the involvement of sucrose transporters in supplying C to the embryo and advocate the idea that manipulating the functions of sucrose transporters within the transfer cells enhances seed nutrition.
Numerous studies have addressed the potential roles of SWEETs at the source and sink levels. 23,105,118,133 The upregulation of the SWEET transporter gene enhanced the fructose to glucose ratio (Fgr) in tomato (Solanum lycopersicum L.), leading to an increased sugar accumulation in fleshy fruits, thereby emphasising the potential contributions of SWEET proteins in sugar accumulation at the sink level. 134 AtSWEET10 135 and its homologue in grapes (Vitis vinifera L.), VvSWEET10, significantly trigger sugar accumulation and transport during fruit ripening. 105 Sugar accumulation in the source leaves reduces the photosynthetic rate and thus affects sink growth. The overexpression of ZjSWEET 2.2 in Jujube led to a drastic reduction in leaf sugar levels and an increase in sucrose import to the sink cells during the fruiting stage, 111 demonstrating the role of the ZjSWEET 2.2 transporter in sugar export from the photosynthesising leaves.
C and N metabolisms are strongly intertwined since amino acid synthesis supports carbohydrate synthesis and distribution. 136,137 Attempts to modify sucrose transport to sink must consider possible interactions with N metabolism and partitioning mechanisms within the source and sink cells. Indeed, the overexpression of  SUT1 resulted in enhanced photosynthesis and C import to the sink organs for supporting amino acid synthesis, transport, and N metabolism in pea. 52 The increased partitioning of C and N in the SUT1-OE seeds resulted in an increment in the yield and improved starch, sucrose, and protein content in seeds. 52 The findings from transgenic pea plants demonstrate the role of SUT1-OE in altering N metabolic pathways in response to sucrose metabolism and allocation with the ultimate goal of improving sink strength and growth in plants. 52 Interestingly, this adaptive adjustment in C and N metabolism and transport processes also applies in reverse situations. 51,138 These findings further support the intricate relationship between sucrose and amino acid metabolic processes in plants. Although the literature has shown a marked increase in the yield in SUTs/SUCs/SWEETs overexpressing plants, less is known about the regulatory mechanisms and pathways mediating sucrose transport.

Contributions of N-responsive genes to carbon and nitrogen distribution processes
N-responsive genes are typically involved in mediating the xylem-phloem transfer of N to the leaves for enhancing photosynthesis, which subsequently promote carbohydrate import to the sink for growth and development. 121,139 Therefore, it becomes imperative to understand the role of N-responsive genes in C and N distribution processes at both the source and the sink levels (refer to Table 2 for further details). Santos-Filho et al. 12 used a NR double-deficient mutant, nia1 nia2, to examine how nitrate assimilation affects the C and N status during the flowering stage in Arabidopsis. The results suggested a significant reduction of NR activity in the leaves, roots, and floral buds of the mutant. Although the reduction in NR activity affected leaf metabolism, the metabolic activities in the sink were found to be unaffected. 12,140 These findings illustrated the contribution of NR in leaf nitrate assimilation and its role in source-sink interactions. Moreover, the nia1 nia2 mutant leaves exhibited a drastic reduction in the amino acid content compared to their wild type, which had a high leaf amino acid content. 12 Previous studies also reported similar findings, 141 suggesting that the roots cannot assimilate enough NH 4 + to meet the required N demand in the leaves. 55 The inadequate leaf N content caused a drastic reduction in chlorophyll content and Rubisco activity, thereby impairing CO 2 assimilation. 12 In contrast, loss of Arabidopsis AMINO ACID PERMEASE2 (AAP2) function markedly increased Rubisco level, photosynthetic rate, and glucose content and upregulated the expression of genes encoding C assimilation. 127 It is possible that the transient glucose accumulation in the vacuoles coupled with the increased expression of hexose importer halted photosynthetic inhibition in the aap2 mutant leaves.
Moreover, the nia1 nia2 mutant leaves exhibited a drastic reduction in total sucrose levels due to the diversion of sucrose to the sink organ for supporting the carbohydrate demand in roots, thus improving NH 4 + assimilation and sink strength. 12 The increased C photoassimilate in roots was associated with the upregulated expression of sucrose transporter in aap2 mutant leaves, resulting in a high C: N ratio in mutant seeds. 127 While roots are the primary sink organs in most plants, the floral bud, which is mainly involved in reproduction, is another principal sink tissue. In Arabidopsis nia1 nia2 mutants, more amino acids were accumulated in the flower buds than in the roots and leaves, despite exhibiting reduced NR activity. 12 These results supported the hypothesis that floral buds prioritise amino acid export from the source leaves over inorganic N assimilates. 142 Less is known about the particular sink branch that accumulates the photoassimilates for development.
In a recent study, a drastic decline in the total amino acid content was observed in the leaf blade of rice DNA BINDING WITH ONE FINGER 11 (Osdof11) mutant, whereas there was an increase in the amino acid content in the seeds. In the Osdof11 mutant, the soluble sugar level was not affected in the leaf blade, while it was drastically reduced in the seeds. 121 OsDOF11 mediated sugar transport by regulating the expression of sucrose transporter genes, OsSUT and OsSWEET. 121 Thus, the reduced sucrose transport observed in the Osdof11 mutant was strongly associated with the drastic reductions in leaf amino acid efficiency and sink size. 121,122 A comprehensive understanding of the combined action of these C-and N-responsive genes could provide a deep insight into the regulation of assimilate transport.

EMPLOYING OMICS TECHNOLOGIES IN CARBON AND NITROGEN METABOLISM: PROSPECT FOR IMPROVED CROP YIELDS
C and N are critical factors that determine plant growth. Therefore, it is crucial to understand the specific compounds that reprogramme the C-N network in the source-sink system. To identify and integrate specific compounds and pathways involved in C and N metabolism, several studies have employed omics approaches, such as metagenomics, transcriptomics, proteomics, and metabolomics. 48,[143][144][145][146][147] The future success of crop research programs depends on identifying and understanding the functions of genes, proteins, and metabolites involved in C and N distribution, and omics techniques have proven to be useful in this regard. In the subsequent sections, we discuss the application of omics approaches in improving crop yield.

Genomics
Recent advances in meta-genomics techniques have provided insightful genomic information and diversity associated with C and N metabolism. 148,149 Numerous studies involving quantitative trait loci (QTL) have been conducted to unravel the C and N metabolism in  various plant species. [150][151][152] These studies have identified specific genetic regions modulating C and N metabolic processes in plants, including nitrogen use efficiency (NUE) and photosynthesis. 48,153,154 It is well known that C 4 grasses are more productive than C 3 grasses, but most of the economically important crops (such as wheat, rice, and potato) utilise C 3 photosynthesis. Several genomics-based efforts have been made to convert these crops to a C 4 system, one of which was conducted by Zhang et al. 48 The study demonstrated that alterations in the expression of genes facilitating C 4 carbon transport are critical for plant adaptability, growth, and yield. The crew also showed that the levels of C and N metabolites are regulated by genetic variations in the key genes encoding C assimilation and transport (malate transporter). 48 These proteins further influence other processes in C 4 photosynthesis including the movement of CO 2 to the mesophyll space and malate transport into the bundle sheath. Manipulating these genetic loci for breeding purposes, followed by an adaptation of these techniques, could promote crop yield than envisaged. In this regard, Cook et al. 153 employed both genomewide associated studies (GWAS) and candidate gene mining techniques to identify key alleles related to C and N metabolism that promote oil, protein, and starch content in maize. Liu et al. 154 performed a GWAS study on 263 maize inbred lines, genotyped with the SNP50 BeadChip maize array. They identified the QTLs, single nucleotide polymorphisms (SNPs), and candidate genes, which may assist in the molecular regulation of kernel starch while improving maize germplasm. 154 A total of 77 identified candidate genes were strongly associated with starch synthesis, suggesting their role in regulating kernel starch content and maize germplasm improvement. 154 These candidate genes appear to be promising in improving plant C and N metabolic processes and ultimately crop improvement. Previous studies have majorly focussed on the identifying loci linked to these traits via linkage mapping or association mapping across generations of unrelated plant species. However, the genetic variations of C and N metabolic processes have been less explored to date, especially in cotton. Recent advances targeted at improving C and N metabolic processes have also exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system for genome editing in diverse crop species. [155][156][157] Ding et al. 158 reported how alterations in the activities of individual enzymes, such as sedoheptulose-1,7-bisphosphatase (SBPase) affect photosynthesis, carbohydrate accumulation, protein synthesis, and amino acid contents in tomato. These metabolic changes are geared towards improving carbon and nitrogen distribution, a prerequisite for improved crop yields.

Transcriptomics
C-and N-responsive pathways are intricately connected, emphasising the need for transcriptome analysis for improving yields. Transcriptomics helps identify genes, transcription factors, and metabolic pathways involved in C and N sensing and signalling. 159 Comparative gene expression analysis has revealed several functionally related C-and Nresponsive genes expressed mainly in aerial parts that support rice growth. 159 Microarray analysis was used by Palenchar et al. 160 to determine the changes in mRNA levels and showed that over 300 genes were downregulated in the combined C:N treated group, providing in vivo evidence of a C/N regulatory pathway. Microarray analysis has also been performed to unravel the N-responsive genes. 161 Although the microarray technique has been used for decades, RNA sequencing (RNA-Seq) is a modern technique that can accurately quantify alterations in the expression of Cand N-responsive metabolic genes. 162 RNA-seq analysis was conducted in rice 163 and cotton 164 to identify differentially expressed genes associated with C and N metabolism in response to N supply. Iqbal et al. 164 employed illumina-based sequencing to examine the genetic variation in cotton in response to N-starvation and resupply. They identified about 75 and 33 hub genes in the shoot and root of cotton involved in C-N metabolism, suggesting N-efficient cotton genotypes for breeders. Besides whole-genome RNA-seq, single-cell RNA seq analysis has been performed to uncover gene expression profiles in small subpopulations of plant cells and molecular interactions among plant cells involved in C and N distribution. 165 A recent study utilised single-cell RNA-seq to identify N-metabolic genes involved in chlorophyll synthesis (a key determinant of photosynthetic capacity and amino acid synthesis) and root development. 166 Overall, these findings demonstrate that identifying C-and N-responsive genes and pathways is a critical step towards achieving worldanticipated yield prospects.

Proteomics
Transcriptome analysis has identified various stressresponsive genes and pathways involved in the intricated C-N network in plants including cotton. 159,164 However, mRNA levels alone are not accurate indicators of gene expression due to post-translational modifications. Therefore, relying solely on genome and transcriptome data is insufficient to determine the gene function and the plant regulatory network in response to stress. 167 To gain a more comprehensive understanding, profiling experiments with translated mRNA might reveal the importance of proteomics. Proteomics aids to identifying the detailed function of differentially expressed proteins in response to stress. 168,169 It explains the mechanisms of plant stress response and growth dynamics, thereby promoting crop yield and quality. Proteomics-based identification of traits related to C and N metabolism could provide molecular interventions to enhance crop performance. Chandna and Ahmad 170 conducted proteomics analysis to identify differentially expressed proteins in wheat in response to N stress, providing an in-depth 68 -MODERN AGRICULTURE understanding of proteins directly associated with photosynthesis, N metabolism, and glycolysis in response to N supply. These findings, along with other studies, 171,172 confirmed the intricated relationship between C and N metabolism at the proteomic level in relation to improved yield. Baslam et al. 173 provide a more detailed review of this subject for further reading.

Metabolomics
Metabolome encompasses all the metabolites present inside the cells, and metabolomics refers to the study of these metabolites. Metabolomics is a systematic approach that identifies changes in the endogenous metabolite levels that perform several physiological functions. 173 Data obtained using this approach can be integrated with proteomics or transcriptomics analyses to identify candidate genes involved in C and N metabolism. A labyrinth of metabolic pathways is involved in C and N coordination. Thus, quantifying metabolite levels using the omics approaches contributes to developing an understanding plant cell responses to environmental or physiological changes induced by C and N status. 174,175 Various metabolomics techniques, including mass spectrometry (MS) and nuclear magnetic resonance (NMR), have successfully measured endogenous metabolites and identified the regulatory networks involved in C and N metabolism. [176][177][178] C and N imbalance is associated with changes in metabolites that affect plant growth and yield. 179, 180 Metabolomics studies have examined the regulation and function of candidate genes involved in the C and N metabolism in various plants tissues (i.e., roots, leaves, grains, and fruits), 181 plant response to phenotypic changes, 174 and plant response to stress. 182,183 Yang et al. 184 revealed that primary metabolites, such as organic acids, amino acids, and sugars, are involved in plant response to magnesium stress. It was observed that assimilates tend to accumulate more at the source leaves and less at the sink organ (root). These responses demonstrated the distinct response of C and N in the leaves and roots of soybean under magnesium deficiency conditions. The integration of transcriptome and metabolome analyses has yielded interesting results and 15 hub genes involved in C and N metabolism under limited N conditions. 163 In addition, synergistic metabolome and proteome analyses have also dissected the underlying molecular mechanism involved in C-and N-related traits. [185][186][187] Integrating these omics technologies could help to identify candidate genes, metabolites, and proteins that regulate C and N distribution in plants.

CONCLUSION AND PERSPECTIVES
Numerous attempts have been made to improve crop performance by modifying C and N partitioning, but achieving our target crop yield remains limited. It is essential not to restrict crop yield improvement solely to C/N balanced distribution but to consider other plant nutrients and their possible impact on the C/N metabolism and the partitioning process. An excellent example is the plant response to changes in C/P balance after the overexpression of SUT in the companion cells. 114 Combining the transport systems of manipulated C, N, and other nutrients while examining their synergetic action could yield deeper insight into their effect on plant growth. Attempts to introduce C and N into the complex network of nutrient assimilation and distribution processes in different plants have been unsuccessful. Therefore, a more holistic approach that compares nutrient partitioning in high-yielding and lowyielding ancestral varieties may provide deeper insight into the complex web of intertwined networks altered in domesticated crop plants.
Synchronizing omics technologies could facilitate the identification of changes in candidate genes, metabolites, and proteins involved in C and N distribution. Recent advances have highlighted the potential of various omics techniques, including glycomics, peptidomics, phenomics, and hormonomics (for review, see Ref. 173). However, there is a paucity of information on how the application of these techniques could improve plant growth via C-N dynamics. Integrating C and N transcriptomics and epigenomics-based research can clarify the effect of changes in gene expression from cellular to whole genome level. 173 Overexpressing sucrose transporters in plants have been shown to alter N metabolism and distribution in response to C assimilate partitioning, thereby improving sink performance. 52,106 Similar findings were observed with N-related genes regulating C and N metabolism in rice. 121,122 With several studies have highlighted the involvement of sucrose and N-responsive genes in C and N metabolism and distribution, less is known about their synergistic action in the distribution process. Understanding this synergistic interaction can help address the following issues: (i) whether the synergistic overexpression of both C-and N-related transporters could simplify transport and metabolism, (ii) how modifications in the C and N distribution influence source capacity and sink strength, and (iii) whether the synergistic action of both transporters can further promote sink growth and strength. In this work, we aim to identify some missing gaps, and a wider integration with other studies and findings focussed on the C and N nutritional network, therefore providing a roadmap for enhancing plant productivity.